Thiosemicarbazates and uses thereof

ABSTRACT

Thioesters, thiocarbamates, thiocarbazates, semithiocarbazates, peptides, aza-amino acid conjugates, and azapeptides; and a chemoselective and site-specific functionalization protocol of protected thiocarbazates and semithiocarbazates are described. The protocol features the use of Mitsunobu reaction to alkylate specifically the nitrogen atom close to the acylthiol moiety with alcohols to produce protected mono-substituted thiocarbazates that can be stored for months, activated under mild conditions at low temperature using halonium reagents and integrated orthogonally to make substituted semicarbazides that can be used, e.g., as synthons in synthesis of aza-amino acid conjugates, azapeptides and other peptidomimetics. Methods for preparing and using ureases, carbazides, semicarbazides, beta-peptides, azapeptides, and other peptidomimetics and azapeptide conjugates, and uses of ureases, carbazides, semicarbazides, beta-peptides, azapeptides in drug discovery, diagnosis, inhibition, prevention and treatment of diseases are also described.

This application claims the benefit of U.S. Provisional Application No.62/845,694, filed on May 9, 2019, hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention is directed to compounds for use in synthesis ofaza-amino acid conjugates, azapeptides, peptoids, azapeptoids, and otherpeptidomimetics; processes for preparing ureases, thioesters,thiocarbamates, thiocarbazates, thiosemicarbazides (collectively“acylthiols”), beta-peptides, and azapepeptides; aza-amino acidconjugates, azapeptides, beta-peptides and other peptidomimeticsprepared from the acylthiols; and uses of the aza-amino acid conjugates,azapeptides, beta-peptides and other peptidomimetics in drug discovery,diagnosis, prevention, inhibition, and treatment of diseases.

BACKGROUND OF THE INVENTION

The in vitro and in vivo stability and in vitro and in vivo half-livesof peptides are limited, e.g., by their rate of hydrolysis and enzymaticdegradation.

Azapeptides are analogs of peptides. An azapeptide contains asubstituted semicarbazide instead of one or more of the amino acidresidue(s) of the parent peptide. In other words, one or more ofα-carbon atom(s) of the parent peptide are replaced with a nitrogen atomin the azapeptide. Due to the reduced reactivity of the carbonyl moietyin the aza-amino acid residue relative to a natural amino acidcounterpart, an aza-peptide bond is more stable under the effect ofpeptidases. Thus, azapeptides are hydrolysed and degraded by peptidasesat a slower rate and exhibit, e.g., an improved metabolic stability,than the parent peptides.

The rate of formation of the aza-peptide bond is much slower than thatof a typical peptide bond. Consequently, there is a greater potential offormation of unwanted side products during azapeptide synthesis withaza-amino acids than with conventional amino acids. An additionalobstacle in utilizing aza-amino acids in syntheses of azapeptides is theorthogonal functionalization of the two available nitrogen atoms in thehydrazine system, peptidomimetics azapeptides with aza-amino acids andconventional coupling agents was challenging prior to the presentinvention.

There is a need for compounds which overcome the limitations ofconventional aza-amino acids and/or allow for a faster, cheaper and moreefficient synthesis of azapeptides, aza-amino acid conjugates and otherpeptidomimetics.

SUMMARY OF THE INVENTION

It is an object of the invention to provide compounds for synthesis ofaza-amino acid conjugates, azapeptides and other peptidomimetics.

It is another object of the invention to provide aza-amino acidconjugates, azapeptides and other peptidomimetics that are more stableand more efficacious than their parent peptides.

It is yet an additional object of the invention to provide azapeptidediagnostic and therapeutic agents.

It is also an object of the invention to provide new and efficaciousmeans and processes for the amide bond formation and coupling of aminoacids, aza-amino acids, aza-amino acid conjugates, peptides, andaza-peptides.

In connection with the above objects and others, the invention providesthiocarbazates, thiosemicarbazates, thiocarbamates and thioesters(collectively, “acylthiols”) and uses of these acylthiols in preparationof various peptides, aza-amino acid conjugates, azapeptides, and otherpeptidomimetics. These acylthiols are stable entities and, prior to use,can be stored for extended periods of time without being compromised. Ascompared to the conventional amino acids and aza-amino acids, theacylthiols could be activated under milder conditions and at lowertemperatures using halonium reagents (e.g., trichloroisocyanuric acid(“TCCA), or a combination of tetrabutyl ammonium chloride (“TBACl”) withtetrabutyl ammonium chloride (“TBACl”)). TBACl enhances reactionperformance when added prior to TCCA. These milder conditions andtemperatures are compatible with, e.g., the protecting groups that arecommonly used in the synthesis of amino acids, aza-amino acids,aza-amino acid conjugates, peptides, azapeptides, and side chains ofconventional amino acids. Accordingly, the acylthiols could beefficiently and practically activated and orthogonally integrated intosemicarbazides, azapeptides and other peptidomimetics and used toprepare aza-amino acid conjugates, both during solution and solid-phaseprotein syntheses. Thus, in certain embodiments, acylthiols are used asbuilding blocks or synthons in synthesises of aza-amino acid conjugates,semicarbazides, azapeptides, and other peptidomimetics. The use of theacylthiols allows, e.g., for the synthesis of aza-amino acid conjugates,semicarbazides, azapeptides, and other peptidomimetics at greater yieldsand/or faster times and/or higher purity, as compared to syntheses ofthese compounds from natural amino acids and aza-amino acids.

The invention also provides a chemoselective and site-specificfunctionalization protocol for protected thiocarbazates andsemithiocarbazates. The protocol features the use of Mitsunobu reactionto alkylate specifically the nitrogen atom close to the acylthiol moietywith alcohols to produce protected mono-substituted thiocarbazates thatare stable and can be stored for prolonged periods of times (e.g.,months) without being compromised. These protected mono-substitutedthiocarbazates can be practically activated under mild conditions usinghalonium reagents (e.g., TCCA or a mixture of TCCA and TBACl) at lowtemperatures, and integrated orthogonally to make substitutedsemicarbazides. The substituted semicarbazides can then be used asbuilding blocks or synthons in the preparation of aza-amino acidconjugates, azapeptides and other peptidomimetics. The protocol iscompatible with the side chains of commonly used amino acids andprotecting groups used in peptide syntheses and, consequently, issuitable for the synthesis of substituted chiral urease and the chiralbeta-amino peptides from the corresponding thiocarbamates and thioesterrespectively, e.g., without epimerization, both in solution phase andsolid-phase protein synthesis.

The invention further provides semicarbazides, azapeptides, azapeptideconjugates, aza-amino acid conjugates and other peptidomimetics preparedfrom the acylthiols and/or by the chemoselective and site-specificfunctionalization protocol.

Thus, in one aspect, the invention provides unsubstituted andsubstituted S-alkylthiocarbazates that can be used in synthesis ofaza-amino acid conjugates, substituted and unsubstituted semicarbazides,azapeptides and other peptidomimetics.

The invention further provides substituted and unsubstitutedsemicarbazides and semithiocarbazides that can be used in synthesis ofaza-amino acid conjugates, azapeptides and other peptidomimetics.

In one aspect, the invention is directed to compounds of Formula (I):

wherein A is selected from the group consisting of H, substituted orunsubstituted branched and unbranched alkyls, substituted orunsubstituted aryls, substituted or unsubstituted heteroaryls, peptides,azapeptides, phthaloyl (Phth), N-phthalimidyl, tert-butoxycarbonyl(Boc), 9-fluorenylmethoxycarbonyl (Fmoc), carboxybenzyl (Cbz),2-(3,5-dimethoxyphenyl)propan-2-yloxycarbonyl (Ddz),2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl (Pbf), trityl ortriphenylmethyl (Trt), t-butyl ester (OtBu), t-butyl ether (tBu),allyloxycarbonyl (Aloc), methoxytrimethylbenzene sulfonyl (Mtr),4,4-dimethyloxybenzhydryl (Mbh),2,2,5,7,8-pentamethyl-chroman-6-sulfonyl chloride (Pmc),2,4,6-trimethoxybenzyl (Tmob), allyl ester (OAI), and acetamidomethyl(Acm);

Y is selected from the group consisting of a bond, NR₁, CHR₂, CHR₃CONR₄,and side chain radicals of amino acids;

X is selected from the group consisting of a bond, NR₅ or CH₂;

D is an H, Cl, alkyl, an aryl or heteroaryl,

R₁, R₂, R₃, R₄, and R₅, is each independently selected from the groupconsisting of a bond, H, alkyls, alkoxyls, alkyl ether, or a protectedalkyl amine. The alkyl, the aryl and heteroaryl may each be substitutedor unsubstituted. In some embodiments, alkyl is selected from the groupconsisting of side chain radicals of amino acids (e.g., aspartic acid,phenylalanine, alanine, histidine, glutamic acid, tryptophan, valine,leucine, lysine, methionine, tyrosine, isoleucine (including,R-isoleucine, S-isoleucine and RS-isoleucine), arginine, glycine,asparagine, serine, and glutamine. The alkyl, aryl, heteroaryl and sidechain radical may each independently be substituted with one or more ofthe following: a halogen (Cl, F), a C₁-C₆ alkyl (e.g., methyl), —COOR,—COR, methoxyl, ethoxyl, propoxyl, a C₁-C₆ haloalkyl (e.g., achloromethyl, a fluromethyl, etc.) or a protecting group (e.g.,N-phthalimidyl, tert-butoxycarbonyl, 9-fluorenylmethoxycarbonyl,2-(3,5-dimethoxyphenyl)propan-2-yloxycarbonyl, etc.), wherein R isselected from the group consisting of alkyls, alkoxyls, alkyl ethers,and protected alkyl amines.

The invention is also directed to compounds of Formula (II):

wherein A is N-phthalimidyl;

D is H, Cl, an alkyl, an aryl or heteroaryl;

R₈ is H or a side chain radical of an amino acid. The alkyl, the aryland heteroaryl may each independently be substituted or unsubstitutedwith one or more of the following: a halogen (Cl, F), a C₁-C₆ alkyl(e.g., methyl), —COOR, —COR, methoxyl, ethoxyl, propoxyl, a C₁-C₆haloalkyl (e.g., a chloromethyl, a fluromethyl, etc.) or a protectinggroup (e.g., N-phthalimidyl, tert-butoxycarbonyl,9-fluorenylmethoxycarbonyl,2-(3,5-dimethoxyphenyl)propan-2-yloxycarbonyl etc.), wherein R isselected from the group consisting of alkyls, alkoxyls, alkyl ethers,and protected alkyl amines.

The invention is further directed to compounds of Formula (MA):

wherein R₁ and R₂ is each independently selected from the groupconsisting of H, halogens, OH, NR₃R₄, alkyls, aryls, and heteroaryls;

R₃ and R₄ is each independently selected from the group consisting of abond, H, alkyls, alkoxyls, alkyl ether, or a protected alkyl amine; and

n is 1, 2, 3, or 4. R₁, R₂, R₃ and R₄ may each independently beunsubstituted or substituted with one or more of the following: ahalogen (Cl, F), a C₁-C₆ alkyl (e.g., methyl), —COOR, —COR, methoxyl,ethoxyl, propoxyl, a C₁-C₆ haloalkyl (e.g., a chloromethyl, afluromethyl, etc.) or a protecting group (e.g., N-phthalimidyl,tert-butoxycarbonyl, 9-fluorenylmethoxycarbonyl,2-(3,5-dimethoxyphenyl)propan-2-yloxycarbonyl, etc.).

The invention is further directed to compounds of Formula (IIIB):

wherein R₁ and R₂ is each independently selected from a group consistingof H, halogens, OH, NR₃R₄, alkyls, aryls, and heteroaryls;

R₃ and R₄ is each independently selected from the group consisting of abond, H, alkyls, alkoxyls, alkyl ether, or a protected alkyl amine;

Ri is selected from a group consisting of halogens (e.g., Cl, F), aC₁-C₆ alkyl (e.g., methyl), —COOR, —COR, methoxyl, ethoxyl, propoxyl, aC₁-C₆ haloalkyl (e.g., a chloromethyl, a fluromethyl, etc.) or aprotecting group (e.g., N-phthalimidyl, tert-butoxycarbonyl,9-fluorenylmethoxycarbonyl,2-(3,5-dimethoxyphenyl)propan-2-yloxycarbonyl, etc.);

R is selected from the group consisting of alkyls, alkoxyls, alkylethers, and protected alkyl amines;

and n is 1, 2, 3, or 4. R₁, R₂, R₃ and R₄ may each be unsubstituted orindependently substituted with one or more of the following: a halogen(Cl, F), a C₁-C₆ alkyl (e.g., methyl), —COOR, —COR, methoxyl, ethoxyl,propoxyl, a C₁-C₆ haloalkyl (e.g., a chloromethyl, a fluromethyl, etc.)or a protecting group (e.g., N-phthalimidyl, tert-butoxycarbonyl,9-fluorenylmethoxycarbonyl,2-(3,5-dimethoxyphenyl)propan-2-yloxycarbonyl, etc.).

The invention is further directed to compounds of Formula (III):

wherein A is a carbamate based protecting group selected from the groupconsisting of tert-butoxycarbonyl, 9-fluorenylmethoxycarbonyl,carboxybenzyl; and

D is H, Cl, an alkyl, an aryl or heteroaryl.

In an additional aspect, the invention is directed to compounds ofFormula (IV):

wherein A is selected from the group consisting of tert-butoxycarbonyl,9-fluorenylmethoxycarbonyl, carboxybenzyl,2-(3,5-dimethoxyphenyl)propan-2-yloxycarbonyl,2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl, trityl ortriphenylmethyl, t-butyl ester, t-butyl ether, allyloxycarbonyl,methoxytrimethylbenzene sulfonyl, 4,4-dimethyloxybenzhydryl,2,2,5,7,8-pentamethyl-chroman-6-sulfonyl chloride,2,4,6-trimethoxybenzyl, allyl ester, and acetamidomethyl;

D is H, Cl, an unsubstituted or substituted alkyl, an unsubstituted orsubstituted aryl or an unsubstituted or substituted heteroaryl;

R₉ is H, alkyl, alkoxyl, alkyl ether, or a protected alkyl amine. Incertain embodiments, R₉ is an alkyl selected from the group consistingof side chain radicals of amino acids (e.g., aspartic acid,phenylalanine, alanine, histidine, glutamic acid, tryptophan, valine,leucine, lysine, methionine, tyrosine, isoleucine (including,R-isoleucine, S-isoleucine and RS-isoleucine), arginine, glycine,asparagine, serine, and glutamine. The alkyl, aryl, heteroaryl, and sidechain radicals may independently be unsubstituted or substituted withone or more of the following: a halogen (Cl, F), a C₁-C₆ alkyl (e.g.,methyl), methoxyl, ethoxyl, propoxyl, a C₁-C₆ haloalkyl (e.g., achloromethyl, a fluromethyl, etc.) or a protecting group, e.g., (e.g.,N-phthalimidyl, tert-butoxycarbonyl, 9-fluorenylmethoxycarbonyl,2-(3,5-dimethoxyphenyl)propan-2-yloxycarbonyletc.).

The invention is also directed to compounds of Formula (V):

wherein A is selected from the group consisting of tert-butoxycarbonyl,9-fluorenylmethoxycarbonyl, carboxybenzyl,2-(3,5-dimethoxyphenyl)propan-2-yloxycarbonyl,2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl, trityl ortriphenylmethyl, t-butyl ester, t-butyl ether, allyloxycarbonyl,methoxytrimethylbenzene sulfonyl, 4,4-dimethyloxybenzhydryl,2,2,5,7,8-pentamethyl-chroman-6-sulfonyl chloride,2,4,6-trimethoxybenzyl, allyl ester, and acetamidomethyl;

D is H, Cl, an unsubstituted or substituted alkyl, an unsubstituted orsubstituted aryl or an unsubstituted or substituted heteroaryl;

R₁₀ is H, alkyl, alkoxyl, alkyl ether, or a protected alkyl amine. Incertain embodiments, R₁₀ is an alkyl selected from the group consistingof side chain radicals of amino acids (e.g., aspartic acid,phenylalanine, alanine, histidine, glutamic acid, tryptophan, valine,leucine, lysine, methionine, tyrosine, isoleucine (including,R-isoleucine, S-isoleucine and RS-isoleucine), arginine, glycine,asparagine, serine, and glutamine. The alkyl, aryl, heteroaryl mayindependently be unsubstituted or substituted with one or more of thefollowing: a halogen (Cl, F), a C₁-C₆ alkyl (e.g., methyl), —COOR,methoxyl, ethoxyl, propoxyl, a C₁-C₆ haloalkyl (e.g., —CF₃, CHF₂, CH₂F,—CCl₃, —CHCl₂, —CH₂Cl)) or a protecting group (e.g., N-phthalimidyl,tert-butoxycarbonyl, 9-fluorenylmethoxycarbonyl,2-(3,5-dimethoxyphenyl)propan-2-yloxycarbonyl, etc.), wherein R isselected from the group consisting of alkyls, alkoxyls, alkyl ethers,and protected alkyl amines.

The invention is further directed to compounds of Formula (VI):

wherein A is selected from the group consisting of N-phthalimidyl,tert-butoxycarbonyl, 9-fluorenylmethoxycarbonyl, carboxybenzyl,2-(3,5-dimethoxyphenyl)propan-2-yloxycarbonyl,2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl, trityl or triphenylmethyl, t-butyl ester, t-butyl ether, allyloxycarbonyl,methoxytrimethylbenzene sulfonyl, 4,4-dimethyloxybenzhydryl,2,2,5,7,8-pentamethyl-chroman-6-sulfonyl chloride,2,4,6-trimethoxybenzyl, allyl ester, and acetamidomethyl;

D is H, Cl, an unsubstituted or substituted alkyl, an unsubstituted orsubstituted aryl, or an unsubstituted or substituted heteroaryl; and

R₁₁ is H, alkyl, aryl, heteroaryl, lipid, peptide, or a side chainradical of an amino acid.

The invention is also directed to compounds of Formula (VII):

wherein A is selected from the group consisting of peptides,azapeptides, N-phthalimidyl, tert-butoxycarbonyl,9-fluorenylmethoxycarbonyl, carboxybenzyl,2-(3,5-dimethoxyphenyl)propan-2-yloxycarbonyl,2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl, trityl or triphenylmethyl, t-butyl ester, t-butyl ether, allyloxycarbonyl,methoxytrimethylbenzene sulfonyl, 4,4-dimethyloxybenzhydryl,2,2,5,7,8-pentamethyl-chroman-6-sulfonyl chloride,2,4,6-trimethoxybenzyl, allyl ester, and acetamidomethyl;

D is H, Cl, an unsubstituted or substituted alkyl, an unsubstituted orsubstituted aryl or an unsubstituted or substituted heteroaryl; and

R₁₂ is H, alkyl, aryl, heteroaryl, lipid, or a side chain radical of anamino acid.

The invention is also directed to compounds of Formula (VIII):

wherein A is selected from the group consisting of peptides,azapeptides, phthalimidyl, tert-butoxycarbonyl,9-fluorenylmethoxycarbonyl, carboxybenzyl,2-(3,5-dimethoxyphenyl)propan-2-yloxycarbonyl,2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl, trityl or triphenylmethyl, t-butyl ester, t-butyl ether, allyloxycarbonyl,methoxytrimethylbenzene sulfonyl, 4,4-dimethyloxybenzhydryl,2,2,5,7,8-pentamethyl-chroman-6-sulfonyl chloride,2,4,6-trimethoxybenzyl, allyl ester, and acetamidomethyl;

D is H, Cl, an unsubstituted or substituted alkyl, unsubstituted orsubstituted aryl, or an unsubstituted or substituted heteroaryl; and R₁₃is H or a side chain radical of an amino acid.

The invention is also directed to compounds of Formula (IX):

wherein A is selected from the group consisting of peptides,azapeptides, N-phthalimidyl, tert-butoxycarbonyl,9-fluorenylmethoxycarbonyl, carboxybenzyl,2-(3,5-dimethoxyphenyl)propan-2-yloxycarbonyl,2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl, trityl ortriphenylmethyl, t-butyl ester, t-butyl ether, allyloxycarbonyl,methoxytrimethylbenzene sulfonyl, 4,4-dimethyloxybenzhydryl,2,2,5,7,8-pentamethyl-chroman-6-sulfonyl chloride,2,4,6-trimethoxybenzyl, allyl ester, and acetamidomethyl;

D is H, Cl, an unsubstituted or substituted alkyl, an unsubstituted orsubstituted aryl, or an unsubstituted or substituted heteroaryl; and

R₁₄ and R₁₅ is each independently selected from the group consisting ofa bond, H, alkyls, alkoxyls, and side chain radicals of amino acids.

The invention is also directed to compounds of Formula (X):

wherein A is phthaloyl, tert-butoxycarbonyl, 9-fluorenylmethoxycarbonyl,carboxybenzyl, 2-(3,5-dimethoxyphenyl)propan-2-yloxycarbonyl,2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl, trityl ortriphenylmethyl, t-butyl ester, t-butyl ether, allyloxycarbonyl,methoxytrimethylbenzene sulfonyl, 4,4-dimethyloxybenzhydryl,2,2,5,7,8-pentamethyl-chroman-6-sulfonyl chloride,2,4,6-trimethoxybenzyl, allyl ester, and acetamidomethyl;

Z is O or S;

R₁₆ and R₁₇ is each independently a bond, H, an alkyl, alkoxyls, or aside chain radicals of an amino acid; and

D is H or an unsubstituted or substituted alkyl, an unsubstituted orsubstituted aryl, an unsubstituted or substituted heteroaryl,phthalimidyl, tert-butoxycarbonyl, 9-fluorenylmethoxycarbonyl,carboxybenzyl, 2-(3,5-dimethoxyphenyl)propan-2-yloxycarbonyl,2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl, trityl ortriphenylmethyl, t-butyl ester, t-butyl ether, allyloxycarbonyl,methoxytrimethylbenzene sulfonyl, 4,4-dimethyloxybenzhydryl,2,2,5,7,8-pentamethyl-chroman-6-sulfonyl chloride,2,4,6-trimethoxybenzyl, allyl ester, or acetamidomethyl. In certainembodiments, Z-D is replaced with a moiety selected from the groupconsisting of —NH₂, —NHR₁, or —NR₂R₃, wherein R₁, R₂ and R₃ is eachindependently selected from the group consisting of alkyls, alkoxyls,alkyl ether, or protected alkyl amines.

The invention is also directed to compounds of Formula (XI):

wherein A is phthaloyl, phthalimidyl, tert-butoxycarbonyl,9-fluorenylmethoxycarbonyl, carboxybenzyl,2-(3,5-dimethoxyphenyl)propan-2-yloxycarbonyl,2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl, trityl ortriphenylmethyl, t-butyl ester, t-butyl ether, allyloxycarbonyl,methoxytrimethylbenzene sulfonyl, 4,4-dimethyloxybenzhydryl,2,2,5,7,8-pentamethyl-chroman-6-sulfonyl chloride,2,4,6-trimethoxybenzyl, allyl, ester, and acetamidomethyl;

R₁₈ is a bond, H, an alkyl, an alkoxyl, an alkylalkoxyl, or a side chainradicals of an amino acid; and

D is H or an unsubstituted or substituted alkyl, an unsubstituted orsubstituted aryl, or an unsubstituted or substituted heteroaryl. Incertain embodiments, OB is —NH₂, —NHR₁, or —NR₂R₃, wherein R₁, R₂ and R₃is each independently selected from the group consisting of alkyls,alkoxyls, alkyl ether, or protected alkyl amines.

In addition, the invention is also directed to compounds of Formula(XII):

wherein A is N-phthalimidyl, tert-butoxycarbonyl,9-fluorenylmethoxycarbonyl, carboxybenzyl,2-(3,5-dimethoxyphenyl)propan-2-yloxycarbonyl,2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl, trityl ortriphenylmethyl, t-butyl ester, t-butyl ether, allyloxycarbonyl,methoxytrimethylbenzene sulfonyl, 4,4-dimethyloxybenzhydryl,2,2,5,7,8-pentamethyl-chroman-6-sulfonyl chloride,2,4,6-trimethoxybenzyl, allyl ester, and acetamidomethyl;

R₁₉ and R₂₀ is each independently a bond, H, an alkyl, an alkoxyl, analkylalkoxyl, an alkylaryloxyl, or a side chain radicals of an aminoacid; and

D is H or an unsubstituted or substituted alkyl, an unsubstituted orsubstituted aryl, or an unsubstituted or substituted heteroaryl. Incertain embodiments, OB is —NH₂, —NHR₁, or —NR₂R₃, wherein R₁, R₂ and R₃is each independently selected from the group consisting of alkyls,alkoxyls, alkyl ether, or protected alkyl amines.

The invention is further directed to compounds of Formula (XIII):

wherein A is selected from the group consisting of peptides,azapeptides, phthaloyl, tert-butoxycarbonyl, 9-fluorenylmethoxycarbonyl,carboxybenzyl, 2-(3,5-dimethoxyphenyl)propan-2-yloxycarbonyl,2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl, trityl ortriphenylmethyl, t-butyl ester, t-butyl ether, allyloxycarbonyl,methoxytrimethylbenzene sulfonyl, 4,4-dimethyloxybenzhydryl,2,2,5,7,8-pentamethyl-chroman-6-sulfonyl chloride,2,4,6-trimethoxybenzyl, allyl ester, and acetamidomethyl;

Y is selected from the group consisting of a bond, —NR₁₆, —CHR₁₇, andside chain radicals of amino acids;

X is selected from the group consisting of a bond, —NR₁₈ and —CHR₁₉;

K is a halogen; and

R₁₅, R₁₆, R₁₇ and R₁₈ is each independently selected from the groupconsisting of a bond, H, alkyls, alkoxyls, and side chain radicals ofamino acids.

The invention is also directed to compounds of Formula (XIV):

wherein A is phthalimidyl;

K is a halogen (e.g., chloride); and

R₁₉ is H or a side chain radical of an amino acid.

The invention is further directed to compounds of Formula (XV):

wherein A is selected from the group consisting of, tert-butoxycarbonyl,9-fluorenylmethoxycarbonyl, carboxybenzyl, and2-(3,5-dimethoxyphenyl)propan-2-yloxycarbonyl; and

K is a halogen (e.g., chloride).

In certain embodiments, A of compounds of Formulas (I)-(XV) is selectedfrom the group consisting of, tert-butoxycarbonyl,9-fluorenylmethoxycarbonyl, carboxybenzyl.

In some embodiments, B of compounds of Formulas (I) to (X) is anunsubstituted or substituted alkyl, an unsubstituted or substitutedaryl, or an unsubstituted or substituted heteroaryl. In someembodiments, B is of compounds of Formulas (I) to (X) is anunsubstituted or substituted alkyl. In some embodiments, theunsubstituted or substituted alkyl is a branched or unbranched C₂-C₂₆alkyl.

In certain embodiments, the alkyl of compounds of Formulas (I)-(XV) is aC₁-C₉ alkyl. In some of these embodiments, the alkyl is selected fromthe group consisting of methyl, ethyl, propyl, isopropyl, andtert-butyl. In some of these embodiments, the alkyl is ethyl.

R₁ to R₁₈ of compounds of Formulas (I)-(XV) could each be independentlyselected from the group consisting of side chain radicals of asparticacid, phenylalanine, alanine, histidine, glutamic acid, tryptophan,valine, leucine, lysine, methionine, tyrosine, isoleucine (including,R-isoleucine, S-isoleucine and RS-isoleucine), arginine, glycine,asparagine, serine, and glutamine.

In certain embodiments, B of compounds of Formula (I)-(XII), is selectedfrom an alkyl, phthalimidyl, tert-butoxycarbonyl,9-fluorenylmethoxycarbonyl, and carboxybenzyl. In some of theseembodiments, B is phthalimidyl (Phth).

In certain embodiments, compounds of Formula (I or II) are selected fromthe group consisting of:

and pharmaceutically acceptable salts thereof, wherein “PG” is H or aprotecting group. The protecting group could, e.g., be N-phthalimidyl,tert-butoxycarbonyl, 9-fluorenylmethoxycarbonyl, or2-(3,5-dimethoxyphenyl)propan-2-yloxycarbonyl.

In certain embodiments, compounds of Formula (I or II) are selected fromthe group consisting of tert-butylN-(1,3-dioxoisoindolin-2-yl)-N-((ethylthio)carbonyl)glycinate(N-Phthaloyl Aza-t-butyl aspartate S-ethyl ester), S-ethylbenzyl(1,3-dioxoisoindolin-2-yl)carbamothioate (N-PhthaloylAza-phenylalanine S-ethyl ester), S-ethyl(1,3-dioxoisoindolin-2-yl)(methyl)carbamothioate (N-PhthaloylAza-alanine S-ethyl ester), S-ethyl((1H-imidazol-5-yl)methyl)(1,3-dioxoisoindolin-2-yl)carbamothioate or(N-Phthaloyl Aza-histidine S-ethyl ester),dioxoisoindolin-2-yl)((ethylthio)carbonyl)amino)propanoate or(N-Phthaloyl Aza-(t-butyl Glutamate) S-ethyl ester), S-ethyl((1H-indol-3-yl)methyl)(1,3-dioxoisoindolin-2-yl)carbamothioate(N-Phthaloyl Aza-tryptophan S-ethyl ester), S-ethyl(1,3-dioxoisoindolin-2-yl)(isopropyl)carbamothioate (N-PhthaloylAza-Valine S-ethyl ester), S-ethyl(1,3-dioxoisoindolin-2-yl)(isobutyl)carbamothioate (N-phthaloylAza-Leucine S-ethyl ester), S-ethyl(4-aminobutyl)(1,3-dioxoisoindolin-2-yl)carbamothioate (N-PhthaloylAza-lysine S-Ethyl ester), S-ethyl(1,3-dioxoisoindolin-2-yl)(2-(methylthio)ethyl)carbamothioate(N-Phthaloyl Aza-Methionine S-ethyl ester), S-ethyl(1,3-dioxoisoindolin-2-yl)(4-hydroxybenzyl)carbamothioate(N-PhthaloylAza-tyrosine S-ethyl ester), S-ethylsec-butyl(1,3-dioxoisoindolin-2-yl)carbamothioate (N-PhthaloylAza-isoleucine S-ethyl ester), 5-ethyl(1,3-dioxoisoindolin-2-yl)(3-guanidinopropyl)carbamothioate (N-PhthaloylAza-arginine S-ethyl ester), S-ethyl(1,3-dioxoisoindolin-2-yl)carbamothioate (N-Phthaloyl Aza-GlycineS-ethyl ester), S-ethyl(2-amino-2-oxoethyl)(1,3-dioxoisoindolin-2-yl)carbamothioate(N-Phthaloyl Aza-aspargine S-ethyl ester), and pharmaceuticallyacceptable salts thereof.

In certain embodiments, compounds of Formula (IV) are selected from thegroup consisting of:

wherein “PG” is H or a protecting group. The protecting group could,e.g., be N-phthalimidyl, tert-butoxycarbonyl,9-fluorenylmethoxycarbonyl, or2-(3,5-dimethoxyphenyl)propan-2-yloxycarbonyl.

In certain embodiments, compounds of Formula (IV) are selected from thegroup consisting of N-Phthaloyl-aza-aspartic acyl chloride,N-Phthaloyl-aza-phenylalanine acyl chloride, N-Phthaloyl-aza-alanineacyl chloride, N-Phthaloyl-aza-histidine acyl chloride,N-Phthaloyl-aza-glutamic acyl chloride, N-Phthaloyl-aza-tryptophan acylchloride, N-Phthaloyl-aza-valine acyl chloride, N-Phthaloyl-aza-leucineacyl chloride, N-Phthaloyl-aza-lysine acyl chloride,N-Phthaloyl-aza-cysteine acyl chloride, N-Phthaloyl-aza-tyrosine acylchloride, N-Phthaloyl-aza-leucine acyl chloride,N-Phthaloyl-aza-arginine acyl chloride, N-Phthaloyl-aza-glycine acylchloride, N-Phthaloyl-aza-asparagine acyl chloride, andN-Phthaloyl-aza-glytamine acyl chloride.

The invention also provides a process for preparing compounds accordingto any one of Formulas (I)-(XII), the process comprising reacting asemi-protected hydrazine with a chlorothioformate to form a protectedalkylthio carbonyl hydrazine (an unsubstituted S-thiocarbazate), andalkylating the protected alkylthio carbonyl hydrazine (the unsubstitutedS-thiocarbazate) via Mitsunobu reaction, or direct alkylation with NaHor an alkyl halide to form compounds of Formulas (I)-(XII) (substitutedS-thiocarbazates). The semi-protected hydrazine may be selected, e.g.,from the group consisting of phthalimidyl-hydrazine,tert-butoxycarbonyl-hydrazine, 9-fluorenylmethoxycarbonyl-hydrazine,carboxybenzyl-hydrazine,2-(3,5-dimethoxyphenyl)propan-2-yloxycarbonyl-hydrazine,2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl-hydrazine, trityl ortriphenylmethyl (Trt)-hydrazine, t-butyl ester-hydrazine, t-butylether-hydrazine, StBu-hydrazine, allyloxycarbonyl-hydrazine,methoxytrimethylbenzene sulfonyl-hydrazine, 4,4-dimethyloxybenzhydryl(Mbh)-hydrazine, 2,2,5,7,8-pentamethyl-chroman-6-sulfonylchloride-hydrazine, 2,4,6-trimethoxybenzyl-hydrazine, allylester-hydrazine, and acetamidomethyl-hydrazine. The chlorothioformatemay be, e.g., an alkylchlorothioformate (e.g., ethylchlorothioformate).In some embodiments, the process further comprises a step of isolatingthe compound of Formula (I)-(XII).

The invention further provides a process for selectively alkylating thenitrogen atom closest to the acylthiol moiety of an unsubstitutedalkylthio carbonyl hydrazine (ATCH) (an unsubstitutedS-alkylthiocarbazate) of Formulas (I) to (XII), the process comprisingreacting the unsubstituted alkylthio carbonyl hydrazine (ATCH)(unsubstituted S-alkylthiocarbazates) of Formulas (I) to (XII) with analcohol to form a protected mono-substituted thiocarbazate. The reactionmay, e.g., be a Mitsunobu reaction. The protected mono-substitutedS-thiocarbazates may then be activated with a halonium reagent(s) (e.g.,TCCA or a mixture of TCCA and TBACl) and coupled with an amine to form asemicarbazide, an azapeptide or an aza-peptide conjugate. In certainembodiments, TCCA can be substituted with N-chlorosuccinamide (NCS),Dichloroisocyanuric acid (DCCA), sodium dichlorocyanuric acid, anyN-chloroamide, calcium hypochlorite, or any N-chloroamide.

The NH moiety in amides or carbamates may react with halonium reagents,and, in certain embodiments, addition of TBACl is used to scavenge theexcess of the halonium ions and help to tune the reactivity to beselectively and specifically toward the acylthiol part withoutinterference with the NH amide or NH carbamates. See Synlett, (2),223-226; 2005, Heterocycles, 76(2), 1511-1524; 2008, Synthesis, (11),1171-4; 1994.

The use of TBACl is particularly useful when Boc, Cbz and Fmocprotecting groups, guanidine, aminoguanidine moieties, or other similarmoieties, are present.

The invention further provides processes that are suitable, e.g., forthe synthesis of substituted chiral urease and the chiral beta-aminopeptides from the corresponding thiocarbamates and thioestersrespectively, without epimerization. The process comprises activation ofa compound of Formulas (I)-(XII) with TCCA or TCCA+TBACl and couplingthe activated acylthiol with an amine. The acylthiol could be, e.g., acompound according to any one of Formulas (I) to (XII), and amine couldbe, e.g., an amino ester, an ester of an amino acid, an amino ester ofan aza-amino acid, a peptide, or an aza-peptide.

A process for preparing a substituted semicarbazide may comprise thesteps of activating a compound according to any one of Formulas (I) to(XII) with TCCA and TBCl to form a compound according to any one ofFormulas (XIII) to (XV), and coupling the compound according to any oneof Formulas (XIII) to (XV) with an amine. The amine could be selectedfrom the group consisting of amino esters, esters of amino acids, aminoesters of aza-amino acids, peptides, and aza-peptides. The reaction maybe performed, e.g., in chloroform, dichloromethane, or acetone. In someembodiments, from about 0.5 to about 2 equivalents of TCCA and TBCl areused. In some embodiments, from about 1 to about 3 equivalents of theamine are used. In some embodiments, from about 1.1 to about 1.8equivalents of TCCA and TBCl, and from about 1.0 to about 1.5equivalents of the amine are used. The coupling may, e.g., be for a timeperiod of from 15 minutes to 12 hours. In certain embodiments, thecoupling may be completed in about 30 minutes, about 40 minutes, about50 minutes; about 60 minutes, about 70 minutes, about 80 minutes, about90 minutes, about 100 minutes, about 110 minutes, or about 120 minutes.In certain embodiments, acetonitrile is used as a solvent both duringthe activating and coupling steps. In certain embodiments,dimethylformamide is used as a solvent during the coupling step.

In an additional aspect, the invention is directed to a process forsynthesizing azapeptides comprising activating a thioester with TCCA andTBCl to form a reactive acyl chloride, and coupling the reactive acylchloride with an amine, wherein the thioester is a compound according toany one of Formulas (I)-(XII), the reactive amine chloride is a compoundaccording to any one of Formulas (XIII)-(XV), and), and the amine isselected from the group consisting of amino esters, esters of aminoacids, amino esters of aza-amino acids, peptides, and aza-peptides. Insome of these embodiments the amine is a peptide or an aza-peptide.

The invention is also directed to a process for a systematic insertionof an aza-amino acid or an aza-amino acid segment(s) at a desiredposition(s) along the peptide sequence comprising activating a thioesterwith TCCA and TBCl to form in situ a reactive acyl chloride, andcoupling the reactive chloride with an amine, wherein the thioester is acompound according to any one of Formulas (I)-(XII), and the reactiveamine chloride is a compound according to any one of Formulas(XIII)-(XV). The amine could be selected, e.g., from the groupconsisting of amino esters, esters of amino acids, amino esters ofaza-amino acids, peptides, and aza-peptides.

The invention is further directed to synthesis of a compound of Formula(XVI):

wherein

is at the N-terminus and/or the C-terminus and/or at or adjacent to acleavage and/or a hydrolysis site of the compound of Formula (XVI);

B is selected from the group consisting of hydrogen, —NH₂, —CONH₂,—COOR₁₉, —COOH, —COH, —COC₁-C₄ alkyl, —COC₁-C₄ haloalkyl, —OH, an aminoacid, an aza amino acid, a 2 to 60-mer peptide, a 2 to 60-mer azapeptide, a 2 to 60-mer azatide;

D is selected from the group consisting of —OR₂₀, —OH, —NH₂, —NNH₂,—NHCOCH₃, —NHCH₃, —N(CH₃)₂, —CONH₂, —COOH, —COH, —COC₁-C₄ alkyl,—COC₁-C₄ haloalkyl, an amino acid, an aza amino acid, a 2 to 60-merpeptide, a 2 to 60-mer aza peptide, a 2 to 60-mer azatide;

R₁₉ and R₂₀ is each independently selected from the group consisting ofC₁-C₆ alkyl (e.g., methyl), methoxyl, ethoxyl, propoxyl, a C₁-C₆haloalkyl (e.g., a chloromethyl, a fluromethyl, etc.) or a protectinggroup (e.g., Phth, Boc, Fmoc, Ddz, etc.);

R is selected from the group consisting of side chain radicals ofaspartic acid, phenylalanine, alanine, histidine, glutamic acid,tryptophan, valine, leucine, lysine, methionine, tyrosine, isoleucine(including, R-isoleucine, S-isoleucine and RS-isoleucine), arginine,glycine, asparagine, serine, threonine, cysteine, and glutamine;

the process comprising activating a compound according to any one ofFormulas (I)-(XII) to form a reactive chloride, and coupling thereactive chloride with an amine. The reactive chloride may, e.g., be acompound according to any one of Formulas XIII-XV, and the amine may,e.g., be an amino ester, an ester of an amino acid, an amino ester of anaza-amino acid, a peptide, or an aza-peptide. In certain embodiments,the process is conducted during solid phase synthesis of an azapeptide.In some of these embodiments, R of compound of Formula (XVI) is selectedfrom the group consisting of side chain radicals of aspartic acid,phenylalanine, alanine, histidine, glutamic acid, tryptophan, valine,leucine, lysine, methionine, tyrosine, R-isoleucine, S-isoleucine, andRS-isoleucine, arginine, glycine, asparagine, serine, threonine,cysteine and glutamine. The side chain radical may be unsubstituted orsubstituted with one or more of the following groups: a halogen (Cl, F,or Br), a C₁-C₆ alkyl (e.g., methyl), hydroxyl, —COOH, —COH, methoxyl,ethoxyl, propoxyl, a C₁-C₆ haloalkyl (e.g., a chloromethyl, afluromethyl, etc.) or a protecting group (e.g., Phth, Boc, Fmoc, Ddz,etc.). In some embodiments, B is selected from the group consisting ofhydrogen, —NH₂, —NNH₂, —CONH₂, —COOR₁₉, —COC₁-C₄ alkyl, —COC₁-C₄haloalkyl, —OH, an amino acid, an aza amino acid, a 2 to 60-mer peptide,a 2 to 60-mer aza peptide, a 2 to 60-mer azatide; D is selected from thegroup consisting of —OR₂₀, —NH₂, —NNH₂, —NHCOCH₃, —NHCH₃, —N(CH₃)₂,—CONH₂, —COOH, —COH, —COC₁-C₄ alkyl, —COC₁-C₄ haloalkyl, an amino acid,an aza amino acid, a 2 to 60-mer peptide, a 2 to 60-mer aza peptide, a 2to 60-mer azatide; and R₁₉ and R₂₀ is each independently selected fromthe group consisting of C₁-C₆ alkyl (e.g., methyl), methoxyl, ethoxyl,propoxyl, a C₁-C₆ haloalkyl (e.g., a chloromethyl, a fluromethyl, etc.)or a protecting group (e.g., Phth, Boc, Fmoc, Ddz, etc.); and R isselected from the group consisting of side chain radicals of asparticacid, phenylalanine, alanine, histidine, glutamic acid, tryptophan,valine, leucine, lysine, serine, threonine, methionine, tyrosine,isoleucine (including, R-isoleucine, S-isoleucine and RS-isoleucine),arginine, glycine, asparagine, and glutamine. The side chain radical ofaspartic acid, phenylalanine, alanine, histidine, glutamic acid,tryptophan, valine, leucine, lysine, methionine, tyrosine, isoleucine(including, R-isoleucine, S-isoleucine and RS-isoleucine), arginine,glycine, asparagine, may be unsubstituted or substituted with one ormore of the following: a halogen (Cl, F, or Br), a C₁-C₆ alkyl (e.g.,methyl), hydroxyl, —COOH, —COH, methoxyl, ethoxyl, propoxyl, a C₁-C₆haloalkyl (e.g., a chloromethyl, a fluromethyl, etc.) or a protectinggroup (e.g., Phth, Boc, Fmoc, Ddz). Compounds of Formula (XVI) may beused in drug discovery, diagnosis, prevention, inhibition, and treatmentof diseases. In some embodiment, the process further comprisesdeprotecting the compound of Formula (XVI) with, e.g., hydrazine,piperadine, TFA, acetic acid, thioanisole, EDT, anisole, etc.

The invention is further directed to synthesis of a compound of Formula(XVI):

wherein

is adjacent to the N-terminus and/or the C-terminus of the compound ofFormula (XVI);

B is selected from the group consisting of hydrogen, —NH₂, —CONH₂,—COOR₁₉, —COOH, —COH, —COC₁-C₄ alkyl, —COC₁-C₄ haloalkyl, —OH, an aminoacid, an aza amino acid, a 2 to 60-mer peptide, a 2 to 60-mer azapeptide, a 2 to 60-mer azatide;

D is selected from the group consisting of —OR₂₀, —OH, —NH₂, —NNH₂,—NHCOCH₃, —NHCH₃, —N(CH₃)₂, —CONH₂, —COOH, —COH, —COC₁-C₄ alkyl,—COC₁-C₄ haloalkyl, an amino acid, an aza amino acid, a 2 to 60-merpeptide, a 2 to 60-mer aza peptide, a 2 to 60-mer azatide;

R₁₉ and R₂₀ is each independently selected from the group consisting ofC₁-C₆ alkyl (e.g., methyl), methoxyl, ethoxyl, propoxyl, a C₁-C₆haloalkyl (e.g., a chloromethyl, a fluromethyl, etc.) or a protectinggroup (e.g., Phth, Boc, Fmoc, Ddz, etc.);

R is selected from the group consisting of side chain radicals ofaspartic acid, phenylalanine, alanine, histidine, glutamic acid,tryptophan, valine, leucine, lysine, methionine, tyrosine, isoleucine(including, R-isoleucine, S-isoleucine and RS-isoleucine), arginine,glycine, asparagine, serine, threonine, cysteine, and glutamine; theprocess comprising activating a compound according to any one ofFormulas (I)-(XII) to form a reactive chloride, and coupling thereactive chloride with an amine. The reactive chloride may, e.g., be acompound according to any one of Formulas XIII-XV, and the amine may,e.g., be an amino ester, an ester of an amino acid, an amino ester of anaza-amino acid, a peptide, or an aza-peptide. In certain embodiments,the process is conducted during solid phase synthesis of an azapeptide.In some of these embodiments, R of compound of Formula (XVI) is selectedfrom the group consisting of side chain radicals of aspartic acid,phenylalanine, alanine, histidine, glutamic acid, tryptophan, valine,leucine, lysine, methionine, tyrosine, R-isoleucine, S-isoleucine, andRS-isoleucine, arginine, glycine, asparagine, serine, threonine,cysteine and glutamine. The side chain radical may be unsubstituted orsubstituted with one or more of the following groups: a halogen (Cl, F,or Br), a C₁-C₆ alkyl (e.g., methyl), hydroxyl, —COOH, —COH, methoxyl,ethoxyl, propoxyl, a C₁-C₆ haloalkyl (e.g., a chloromethyl, afluromethyl, etc.) or a protecting group (e.g., Phth, Boc, Fmoc, Ddz,etc.). In some embodiments; B is selected from the group consisting ofhydrogen, —NH₂, —NNH₂, —CONH₂, —COOR₁₉, —COC₁-C₄ alkyl, —COC₁-C₄haloalkyl; —OH, an amino acid, an aza amino acid, a 2 to 60-mer peptide,a 2 to 60-mer aza peptide, a 2 to 60-mer azatide; D is selected from thegroup consisting of —OR₂₀, —NH₂, —NNH₂, —NHCOCH₃, —NHCH₃, —N(CH₃)₂,—CONH₂, —COOH, —COH, —COC₁-C₄ alkyl, —COC₁-C₄ haloalkyl, an amino acid,an aza amino acid, a 2 to 60-mer peptide, a 2 to 60-mer aza peptide, a 2to 60-mer azatide; and R₁₉ and R₂₀ is each independently selected fromthe group consisting of C₁-C₆ alkyl (e.g., methyl), methoxyl, ethoxyl,propoxyl, a C₁-C₆ haloalkyl (e.g., a chloromethyl, a fluromethyl, etc.)or a protecting group (e.g., Phth, Boc, Fmoc, Ddz, etc.); and R isselected from the group consisting of side chain radicals of asparticacid, phenylalanine, alanine, histidine, glutamic acid, tryptophan,valine, leucine, lysine, serine, threonine, methionine, tyrosine,isoleucine (including, R-isoleucine, S-isoleucine and RS-isoleucine),arginine, glycine, asparagine, and glutamine. The side chain radical ofaspartic acid, phenylalanine, alanine, histidine, glutamic acid,tryptophan, valine, leucine, lysine, methionine, tyrosine, isoleucine(including, R-isoleucine, S-isoleucine and RS-isoleucine), arginine,glycine, asparagine, may be unsubstituted or substituted with one ormore of the following: a halogen (Cl, F, or Br), a C₁-C₆ alkyl (e.g.,methyl), hydroxyl, —COOH, —COH, methoxyl, ethoxyl, propoxyl, a C₁-C₆haloalkyl (e.g., a chloromethyl, a fluromethyl, etc.) or a protectinggroup (e.g., Phth, Boc, Fmoc, Ddz). Compounds of Formula (XVI) may beused in drug discovery, diagnosis, prevention, inhibition, and treatmentof diseases. In some embodiment, the process further comprisesdeprotecting the compound of Formula (XVI) with, e.g., hydrazine,piperadine, TFA, acetic acid, thioanisole, EDT, anisole, etc.

The invention is further directed in part to a process of preparing acompound of Formula (XVI) comprising cleaving a peptide at itsN-terminus and/or C-terminus, and coupling the cleaved peptide with acompound according to any one of Formulas (I)-(IX) to form the compoundof Formula (XVI).

The invention is also directed in part to a process of preparing acompound of Formula (XVI) comprising cleaving a peptide at its cleavagesite to form two smaller peptides, replacing the last amino acid of atleast one of the smaller peptides with a compound according to any oneof Formulas (I)-(XII) to form an azapeptide, and conjugating theazapeptide with the remaining smaller peptide to provide a compound ofFormula (XVI).

The invention is also directed in part to a process of preparing acompound of Formula (XVI) comprising hydrolizing a peptide at itscleavage site, and reacting the cleaved peptide with a compoundaccording to any one of Formulas (I)-(XII) to provide a compound ofFormula (XVI).

The invention is further directed in part to a method of azapeptidesynthesis comprising reacting a compound according to any one ofFormulas (I)-(XII) with an aza-amino acid, an amino acid, a peptide, anazapeptide, or an additional compound according to any one of Formulas(I)-(XII) to form an azapeptide. The azapeptide may be, e.g., a compoundof Formula (XVI).

The invention is further directed in part to a solid phase synthesis ofan azapeptide, the solid phase synthesis comprising coupling a compoundaccording to any one of Formulas (I)-(XII) to a support, and coupling anadditional protected compound according to any one of Formulas(I)-(XII), an additional protected amino acid, or an additionalprotected aza-amino acid to the deprotected compound of according to anyone of Formulas (I)-(XII). In certain embodiments, the compoundaccording to any one of Formulas (I)-(XII) may be deprotected prior tosaid coupling.

The invention is further directed in part to a solid phase synthesis ofan azapeptide, the solid phase synthesis comprising coupling a compoundaccording to any one of Formulas (I)-(XII) to a support, deprotectingthe compound according to any one of Formulas (I)-(XII), and couplingthe deprotected compound of according to any one of Formulas (I)-(XII)to a protected compound according to any one of Formulas (I)-(XII), aprotected amino acid, or an a protected aza-amino acid.

The invention is further directed in part to a solution phase synthesisof the compounds of Formula (XVI), the solution phase synthesiscomprising deprotecting a compound of any one of Formulas (I)-(XII), andcoupling the deprotected compound of any one of Formulas (I)-(XII) withan additional compound any one of Formulas (I)-(XII), or a protectedamino acid, or a protected aza-amino acid to form a protectedazapeptide. The synthesis may further comprise a step of deprotectingthe protected azapeptide to provide a compound of Formula (XVI). Thecoupling may, e.g., be for a time period of from about 15 minutes toabout 12 hours. In certain embodiments, the coupling may be completed inabout 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes,about 70 minutes, about 80 minutes, about 90 minutes, about 100 minutes,about 110 minutes, or about 120 minutes.

The synthons and processes of the invention allow, e.g., preparation ofunsubstituted and substituted thiocarbazates, semicarbazides,azapeptides and aza-peptide conjugates, e.g., in yields of at leastabout 50% (by weight) (e.g., from about 55% to about 99%, from about 60%to about 95%, or from about 65% to about 95%). Thus, the yield may,e.g., be about 55%, about 60%; about 65%, about 75%, about 80%, about85%, about 90%, about 95%, about 97%, or about 99%. In certainembodiments, the yield is greater than 85%

Definitions

The term “about” in the present specification means a value within 15%(±15%) of the value recited immediately after the term “about,”including the value equal to the upper limit (i.e., +15%) and the valueequal to the lower limit (i.e., −15%) of this range. For example, thephrase “about 100” encompasses any numeric value that is between 85 and115, including 85 and 115.

An “azatide” means a peptide in which all α-carbons are replaced bynitrogen trivalent atoms.

An “α-nitrogen” means a nitrogen atom bonded to a carbonyl group in anazapeptide or an azatide. The carbon atom next to the α-nitrogen iscalled the β-carbon.

An “aza-amino acid” is defined as an amino acid where the chiralα-carbon atom is replaced by a nitrogen atom.

An “azapeptide analogue” means a compound which differs from a peptidethat it is an analogue of in that one or more α-carbon atoms of thepeptide have been replaced by a nitrogen atom with or without additionalstructural modification(s) to the side chain(s) of the amino acidresidues of the peptide. The one or more α-carbon atoms of the peptidemay, e.g., be at the N-termini of the peptide (i.e., the first residueof the peptide), at the second residue of the peptide, the C-termini ofthe peptide (i.e., the last residue of the peptide), the residuecovalently bound to the C-termini of the peptide, and/or at anotherresidue of the peptide (e.g., at the site of hydrolysis of the peptide).Despite having a backbone different from the peptide, the azapeptideanalogue preserves, extends and/or improves functional activity of thepeptide. The azapeptide analogue is more resistant to degradation thanthe peptide and/or has an improved therapeutic activity than the peptideand/or has an improved selectivity for a biological receptor than thepeptide and/or improved affinity to a biological receptor and/orreversed activity at a biological receptor (agonistic activity insteadof antagonist activity or antagonistic activity instead of agonisticactivity).

An “amine” in the process of the invention may, e.g., be an amino ester,an ester of an amino acid, an amino ester of an aza-amino acid, apeptide, or an aza-peptide, an amino acid, an aza-amino acid, providedthat, if the amino ester, the ester of an amino acid, the amino ester ofthe aza-amino acid, the peptide, the aza-peptide, the amino acid, or theaza-amino acid contains a group selected from amino, amide, guanidino N,carboxyl, sulfhydryl, carboxyl, hydroxyl, indole, imidazole phenol, thegroup is protected with a protecting group selected fromtert-butoxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl (Fmoc), or2-(3,5-dimethoxyphenyl)propan-2-yloxycarbonyl (Ddz), phthalimide (Phth),carboxybenzyl (Cbz), 2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl(Pbf), trityl or triphenylmethyl (Trt), t-butyl ester (OtBu), t-butylether (tBu), allyloxycarbonyl (Aloc), methoxytrimethylbenzene sulfonyl(Mtr), 4,4-dimethyloxybenzhydryl (Mbh),2,2,5,7,8-pentamethyl-chroman-6-sulfonyl chloride (Pmc),2,4,6-trimethoxybenzyl (Tmob), allyl ester (OAT), acetamidomethyl (Acm),and the like. The amino ester may, e.g., be t-butyl, p-methoxy benzylester, glycine ethyl ester, etc.

The term “heteroaryl” includes all aryl compounds with atoms other thanC and H.

The term “protected” as it is used herein means that one or moregroup(s) (e.g., —OH) in an amino acid, an aza-amino acid, a peptide, anazapeptide, or a compound is protected with a protecting group (e.g.,Phth, Boc, Ddz, etc.). Unless otherwise indicated, the term “protectinggroup” or “protective group,” when used to refer to part of a moleculesubjected to a chemical reaction, means a chemical moiety that is notreactive under the conditions of that chemical reaction, and which maybe removed to provide a moiety that is reactive under those conditions.Protecting groups include, for example, nitrogen protecting groups andhydroxy-protecting groups. Examples of protective group include, e.g.,benzyl, diphenylmethyl, trityl, Cbz, Boc, Fmoc, methoxycarbonyl,ethoxycarbonyl, Phth, Ddz, as well as other protective groups known tothose skilled in the art.

The “amino acid side chain radical(s)” in the compounds of Formulas(I)-(XVI) may be a side chain radical of natural amino acid or a sidechain radical of unnatural amino acid. The unnatural amino acidsinclude, e.g., aza-imidazole derivatives and Phth-protected carbamoylaza-benzotriazole derivatives of β-amino acids (e.g., L-β-homotyrosine,β-alanine, L-β-homoasparagine, L-β-homoalanine, L-β-homophenylalanine,L-β-homoproline, L-β-holysine, L-β-homoarginine, L-β-proline, etc.),aliphatic amino acids (e.g., 6-aminohexanoic acid,2-amino-3-methoxybutanoic acid, 1-aminocyclopentane-1-carboxylic acid,2-(aminooxy)acetic acid, 6-aminohexanoic acid,2-[2-(amino)-ethoxy]-ethoxy}acetic acid), β-cyclohexyl-L-alanine,6-aminohexanoic acid, L-α,β-diaminopropionic acid, L-propargylglycinel,L-α,β-diaminopropionic acid, α-aminoisobutyric acid,β-(2-pyridyl)-L-alanine, β-(3-pyridyl)-L-alanine,β-cyclopropyl-L-alanine, β-t-butyl-L-alanine,(2,4-dinitrophenyl))-L-α,β-diaminopropionic acid,(allyloxycarbonyl)-L-α,β-diaminopropionic acid, D-α,β-diaminopropionicacid, L-α,β-diaminopropionic acid,(N-γ-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl)-L-α,γ-diaminobutyricacid, (N-γ-4-methyltrityl)-L-α,γ-diaminobutyric acid,L-α,γ-diaminobutyric acid, 4-fluoro-L-phenylglycine,5,5,5-trifluoro-DL-leucine, epsilon-aminohexanoic-OH,L-α-t-butylglycine, L-2-amino-3-(dimethylamino)propionic acid,L-2-aminocaproic acid, L-allylglycine, lysine azide,(Nδ-4-methyltrityl)-L-ornithine, Arg(Me)(Pbf)-OH, dimethyl-L-arginine(symmetrical and unsymmetrical), L-2-amino-3-guanidinopropionic acid,L-citrulline, ε-acetyl-L-lysine, Lys(ivDde)-OH, Lys(Me)₂-OH.HCl,Lys(Me₃)—OHchloride, α-methyl-DL-glutamic acid, γ-carboxy-L-glutamicacid γ,γ-di-t-butyl ester, (N-γ-ethyl)-L-glutamine, 2,6-diaminopimelicacid, Glu(OAll)-OH, L-cysteic acid, α-methyl-DL-methionine,DL-buthionine, L-cysteic acid, L-selenomethionine,S-[2-(4-pyridyl)ethyl]-L-cysteine, S-[2-(4-pyridyl)ethyl]-L-cysteine,S-diphenylmethyl-L-cysteine, S-trityl-L-homocysteine,S-trityl-L-enicillamine, (Se-p-methoxybenzyl)-L-selenocysteine,β-hydroxyphenylalanine, 2-cyano-L-phenylalanine, L-thyroxine,O-methyl-L-tyrosine, β-methyl-DL-phenylalanine, 2-cyano-L-phenylalanine,L-thyroxine, O-methyl-L-tyrosine, β-methyl-DL-phenylalanine,2-cyano-L-phenylalanine, 3,4-dichloro-L-phenylalanine,3,4-difluoro-L-phenylalanine, 3,4-dihydroxy-L-phenylalanine,3,4-dihydroxy-phenylalanine, 3-amino-L-tyrosine, 3-chloro-L-tyrosine,3-fluoro-DL-tyrosine, 3-nitro-L-tyrosine, 4-amino-L-phenylalanine,4-aminomethyl-L phenylalanine, 4-(phosphonomethyl)-phenylalanine,4-benzoyl-D-phenylalanine, 4-bis(2-chloroethyl)amino-L-phenylalanine,4-cyano-L-phenylalanine, 4-fluoro-L-phenylalanine,4-iodo-L-phenylalanine, DL-m-tyrosine, 2,6-dimethyl-tyrosine,L-homophenylalanine, O-methyl-L-tyrosine, Phe(4-guanidino) OH,O-benzyl-L-phosphotyrosine, (2S,3R)-3-phenylpyrrolidine-2-carboxylicacid, (2S,4S)-4-phenyl-pyrrolidine-2-carboxylic acid,(2S,3aS,7aS)-Octahydro-1H-indole-2-carboxylic acid,(2S,3R)-3-phenylpyrrolidine-2-carboxylic acid, (2S,4R)(−)-4-t-butoxypyrrolidine-2-carboxylic acid, trans-4-Fluoro-L-proline,(3S,4S)-4-amino-3-hydroxy-6-methylheptanoic acid,4-amino-3-hydroxybutanoic acid, L-α-methylserine,(2S,3S)-2-amino-3-methoxybutanoic acid, Thr(β-D-GlcNAc(Ac)3)-OH,O-benzyl-L-phosphoserine, O-benzyl-D-phosphothreonine,O-benzyl-L-phosphothreonine, 4-methyl-DL-tryptophan,6-fluoro-DL-tryptophan, 6-methyl-DL-tryptophan, DL-7-azatryptophan,(R)-7-Azatryptophan, 5-benzyloxy-DL-tryptophan, 5-bromo-DL-tryptophan,5-chloro-DL-tryptophan, 5-fluoro-DL-tryptophan, 5-hydroxy-L-tryptophan,5-methoxy-L-tryptophan, 6-chloro-L-tryptophan, 6-methyl-DL-tryptophan,7-methyl-DL-tryptophan, DL-7-azatryptophan, 5-azido-pentanoic acid,2-Amino-N-(3-azidopropyl)-3-mercaptopropionamide,2-Amino-N-(3-azidopropyl)-3-mercaptopropionamide, Azidohomoalanine,L-propargylglycine-DCHA, azidolysine, p-azidophenylalanine,Azidohomoalanine, D-propargylglycine, L-propargylglycine, azidolysine,Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine, 2-(7′-octenyl)alanine, 2-(4′-pentenyl) alanine, 2-(4′-pentenyl)glycine, 2-(7′-octenyl)alanine, [5-((2-Aminoethyl)amino)naphthalene-1-sulfonic acid],L-glutamic acid-γ-[2-(1-sulfonyl-5-naphthyl)-aminoethylamide],N-ε-(5-carboxyfluorescein)-L-lysine,N-ε-(5/6-carboxyfluorescein)-L-lysine,N-ε-(4,4-dimethylazobenzene-4′carbonyl)-L-lysine,Nε-2,4-dinitrophenyl-L-lysine,N-ε-[(7-methoxycoumarin-4-yl)-acetyl-L-lysine, glycosylated amino acids(e.g., Ser(β-D-GlcNAc(Ac)₃) —OH, Thr(β-D-GlcNAc(Ac)3) —OH),3-azabicyclo[3.1.0]hexane-2-carboxylic acid; 4-amino-(1-carboxymethyl)piperidine, 4-phenylpiperidine-4-carboxylic acid,Nα-methyl-N-im-trityl-L-histidine, Na-methyl-O-benzyl-L-serinedicyclohexylammonium salt,Nalpha-methyl-Nomega-(4-methoxy-2,3,6-trimethylbenzenesulfonyl)-L-arginine,Nalpha-methyl-L-leucine, Nalpha-methyl-L-norvaline,Nalpha-methyl-L-phenylalanine, Nalpha-methyl-N-im-trityl-L-histidine,Nalpha-methyl-O-t-butyl-L-serine, Nalpha-methylglycine,21-amino-4,7,10,13,16,19-hexaoxaheneicosanoic acid,{2-[2-(amino)-ethoxy]-ethoxy}acetic acid, 6-Amino-4-oxohexanoic acid,5-Amino-3-Oxapentamoic Acid, NH-(PEG)10-CH₂CH₂COOH,NH-(PEG)12—CH₂CH₂COOH, 9-Amino-4; 7-Dioxanonanoic acid, 9-Amino-4;7-Dioxanonanoic acid, 12-amino-4,7,10-trioxadodecanoic acid,15-amino-4,7,10,13-tetraoxapentadecacanoic acid,18-amino-4,7,10,13,16-pentaoxaoctadecanoic acid,21-amino-4,7,10,13,16,19-hexaoxaheneicosanoic acid,NH-(PEG)8-CH2CH2COOH, 11-amino-3,6,9-trioxaundecanoic acid,N-(Fmoc-8-amino-3,6-dioxa-octyl)succinamic acid, —N-ε-acetyl-L-lysine,L-citrulline, Arg(Me)(Pbf)-OH, Nω,ω-dimethyl-L-arginine (asymmetricaland symmetrical), Lys(Me)₂-OH chloride, N-ε,ε-t-methyl-L-lysine,Lys(Me3)—OH chloride, O-benzyl-L-phosphoserine,O-benzyl-D-phosphothreonine, O-benzyl-L-phosphothreonine,O-benzyl-L-phosphotyrosine.

A “side chain radical” of aspartic acid, phenylalanine, alanine,histidine, glutamic acid, tryptophan, valine, leucine, lysine,methionine, tyrosine, isoleucine (including, R-isoleucine, S-isoleucineand RS-isoleucine), arginine, glycine, asparagine, and glutamine havethe following structures:

A “side chain radical of proline” is a secondary amine, in that thealpha-amino group is attached directly to the main chain, making the αcarbon a direct substituent of the side chain:

Amino acids which can be used in the present invention include L andD-amino acids.

Unless otherwise indicated, the terms “prevent,” “preventing” and“prevention” contemplate an action that occurs before a patient beginsto suffer from the symptoms of specified disease or disorder, whichinhibits or reduces the severity of the disease or disorder or of one ormore of its symptoms. The terms encompass prophylaxis.

The compounds of the invention can be used in the form ofpharmaceutically acceptable salts derived from inorganic or organicacids. For clarity, the term “pharmaceutically acceptable salt[s]” asused herein generally refers to salts prepared from pharmaceuticallyacceptable acids or bases including inorganic acids and bases andorganic acids and bases. Suitable pharmaceutically acceptable baseaddition salts include, e.g., metallic salts made from aluminum,calcium, lithium, magnesium, potassium, sodium and zinc or organic saltsmade from lysine, N,N′-dibenzylethylenediamine, chloroprocaine, choline,diethanolamine, ethylenediamine, meglumine (N-methylglucamine) andprocaine. Suitable non-toxic acids include inorganic and organic acidssuch as acetic, alginic, anthranilic, benzenesulfonic, benzoic,camphorsulfonic, citric, ethenesulfonic, formic, fumaric, furoic,galacturonic, gluconic, glucuronic, glutamic, glycolic, hydrobromic-,hydrochloric, isethionic, lactic, maleic, malic, mandelic,methanesulfonic, mucic, nitric, pamoic, pantothenic, phenylacetic,phosphoric, propionic, salicylic, stearic, succinic, sulfanilic,sulfuric, tartaric acid, and p-toluenesulfonic acid. Specific acidsinclude, e.g., hydrochloric, hydrobromic, phosphoric, sulfuric, andmethanesulfonic acids. Examples of specific salts include, e.g.,hydrochloride and mesylate salts. Others are well-known in the art. See,e.g., Remington's Pharmaceutical Sciences, 18th ed. (Mack Publishing,Easton Pa.: 1990) and Remington: The Science and Practice of Pharmacy,19th ed. (Mack Publishing, Easton Pa.: 1995). The preparation and use ofacid addition salts, carboxylate salts, amino acid addition salts, andzwitterion salts of compounds of the present invention may also beconsidered pharmaceutically acceptable if they are, within the scope ofsound medical judgment, suitable for use in contact with the tissues ofhumans and lower animals without undue toxicity, irritation, allergicresponse, and the like, are commensurate with a reasonable benefit/riskratio, and are effective for their intended use. Such salts may alsoinclude various solvates and hydrates of the compound of the presentinvention.

Certain compounds of the present invention may be isotopically labelled,e.g., with various isotopes of carbon, fluorine, or iodine, asapplicable when the compound in question contains at least one suchatom. In preferred embodiments, methods of diagnosis of the presentinvention comprise administration of such an isotopically labelledcompound.

Certain compounds of the present invention may exist as stereoisomerswherein, asymmetric or chiral centers are present. These stereoisomersare “R” or “S” depending on the configuration of substituents around thechiral carbon atom. The terms “R” and “S” used herein are configurationsas defined in IUPAC 1974 Recommendations for. Section E, FundamentalStereochemistry, in Pure Appl. Chem., 1976, 45: 13-30. The inventioncontemplates various stereoisomers and mixtures thereof and these arespecifically included within the scope of this invention. Stereoisomersinclude enantiomers and diastereomers, and mixtures of enantiomers ordiastereomers. Individual stereoisomers of compounds of the inventionmay be prepared synthetically from commercially available startingmaterials which contain asymmetric or chiral centers or by preparationof racemic mixtures followed by resolution well known to those ofordinary skill in the art. These methods of resolution are exemplifiedby (1) attachment of a mixture of enantiomers to a chiral auxiliary,separation of the resulting mixture of diastereomers byrecrystallization or chromatography and optional liberation of theoptically pure product from the auxiliary as described in Furniss,Hannaford, Smith, and Tatchell, “Vogel's Textbook of Practical OrganicChemistry”, 5th edition (1989), Longman Scientific & Technical, EssexCM20 2JE, England, or (2) direct separation of the mixture of opticalenantiomers on chiral chromatographic columns or (3) fractionalrecrystallization methods.

Certain compounds of the present invention may exist as cis or transisomers, wherein substituents on a ring may attach in such a manner thatthey are on the same side of the ring (cis) relative to each other, oron opposite sides of the ring relative to each other (trans). Suchmethods are well known to those of ordinary skill in the art, and mayinclude separation of isomers by recrystallization or chromatography. Itshould be understood that the compounds of the invention may possesstautomeric forms, as well as geometric isomers, and that these alsoconstitute an aspect of the invention.

The term “solid-phase synthesis” means a method in which molecules(e.g., amino acids, aza-amino acids, etc.) are covalently bound on asolid support material and synthesised step-by-step in a single reactionvessel utilising selective protecting group chemistry. In this method,building blocks are typically protected at all reactive functionalgroups. The order of functional group reactions can be controlled by theorder of deprotection. For example, in an aza-peptide synthesis, anamino-protected amino acid or an amino-protected aza-amino acid is boundto a solid phase material (e.g., low cross-linked polystyrene beads),forming a covalent bond between the carbonyl group and the resin, e.g.,an amido or an ester bond. Then the amino group is deprotected andreacted with the carbonyl group of the next amino-protected amino acidor amino-protected aza-amino acid. This cycle is repeated to form thedesired peptide or aza-peptide chain. After all reactions are complete,the synthesised peptide or aza-peptide is cleaved from the bead.

The terms “solution phase synthesis” and “liquid phase synthesis” meansa method in which molecules (e.g., amino acids, aza-amino acids, etc.)are synthesized in a solution without being covalently bound on a solidsupport material.

The term “synthon” means a synthetic building block.

The term “room temperature” means 20° C.

The term “ambient temperature” means 18-28° C.

The terms “parent peptide” and “corresponding peptide” mean a nativepeptide (i.e., natural or convention peptide) that differs from anazapeptide in that one or more of the amino residue(s) of the nativepeptide is (are) replaced by a semicarbazide or a substitutedsemicarbazide (i.e., one or more α-carbon(s) of the native peptide arereplaced by nitrogen trivalent atom(s)) in the azapeptide. Thereplacement may be, e.g., at the N-termini of the peptide (i.e., thefirst residue of the peptide), at the second residue of the peptide, theC-termini of the peptide (i.e., the last residue of the peptide), theresidue covalently bound to the C-termini of the peptide, and/or atanother residue of the peptide (e.g., at the site of hydrolysis of thepeptide).

The term “phthalimidyl” means:

The term “phthaloyl” means:

The abbreviation “N-Phth” means “N-phthalimidyl.”

The abbreviation “Boc” means “tert-butoxycarbonyl.”

The abbreviation “Fmoc” means “9-fluorenylmethoxycarbonyl.”

The abbreviation “Ddz” means“2-(3,5-dimethoxyphenyl)propan-2-yloxycarbonyl.”

The abbreviation “HOBt” means “1-OH-Benzotriazole.”

The abbreviation “SPPS” means “Solid Phase Peptide Synthesis.”

The abbreviation “TCCA” means “trichloroisocyanuric acid.”

The abbreviation “TBACl” means “tetrabutyl ammonium chloride.”

The abbreviation “Phth” means “phthaloyl.”

The abbreviation “Cbz” means “carboxybenzyl.”

The abbreviation “Pbf” means“2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl.”

The abbreviation “Trt” means “trityl or, triphenylmethyl.”

The abbreviation “OtBu” means “O-t-butyl.”

The abbreviation “tBu” means “t-butyl.”

The abbreviation “Aloc” means “allyloxycarbonyl.”

The abbreviation “Mtr” means “methoxytrimethylbenzene sulfonyl.”

The abbreviation “Mbh” means “4,4-dimethyloxybenzhydryl.”

The abbreviation “Pmc” means “2,2,5,7,8-pentamethyl-chroman-6-sulfonylchloride.”

The abbreviation “Tmob” means 2,4,6-trimethoxybenzyl.

The abbreviation “OAI” means “allyl ester.”

The abbreviation “Acm” means “acetamidomethyl.”

The abbreviation “DEAD” means “Diethyl Azodicarboxylate.”

The abbreviation “TIPS” means TriIsoPropylSilane.

“Activation” of thiocarbazates or thiocarbamate as used herein meansconverting the thiocarbonyl moiety into active acyl donor. For example,when TCCA/TBACl are used, the anticipated active acyl donor ischloroformate moiter (acyl chloride).

“Coupling” means the event of adding a good nucleophile to the activeacyl donor. For example, it includes formation of an amide, urea, orsemicarbazide from the corresponding acyl chloride.

In peptide chemistry, “deprotection” refers to a process of removing theprotecting groups (e.g., phthaloyl, Boc, Cbz, Fmoc, etc) by a chemicalagent. For example, Boc protecting group could be removed under acidicconditions (e.g., 4 M HCl, or neat trifluoroacetic acid TFA); Fmocprotecting group could be removed under basic conditions when pH ishigher than 12 (20% pipyridine/DMF or DCM); and Phthaloyl group can becleaved, e.g., under basic conditions or by the use of hydrazine.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts X-ray analysis of hydrazine carbonyl chloridechloroformate 35.

FIG. 2 depicts X-ray analysis of substituted ACTH 33 and 34.

FIG. 3 depicts X-ray analysis of A7123.

FIG. 4 depicts X-ray analysis of A790.

FIG. 5 depicts HPLC analysis for 2-azabradykinin made by SPPS.

FIG. 6 depicts HPLC analysis for 8-azabradykinin made by SPPS.

FIG. 7 depicts HPLC analysis for 7-azabradykinin made by SPPS.

FIG. 8 depicts HPLC analysis for 2,8-azabradykinin made by SPPS.

DETAILED DESCRIPTION

The relatively small bond dissociation energies for the α-C—H bonds inamino acids and peptides compared to that in secondary and tertiarystructures make the bond vulnerable to reactive free radicals andoxidants. As a result, some of these peptides are relatively unstableand show very short half-life times.

The exchange of a one or more specific amino acid moiety(ies) in thepeptide sequence with an aza-amino acid produces azapeptides. Thisalteration in the peptide backbone introduces conformational changesbecause of the adjustable chirality on the alpha nitrogen. In addition,it has been suggested that the lone pair on the nitrogen atom plays arole through resonance to favor the β-turn geometry. Resonance in thesemicarbazide moiety also contributes to the reduced electrophilicity ofthe carbonyl system compare to that in typical amide.

Azapeptides exhibit, e.g., an extra stability to hydrolysis byproteases, as compared to the corresponding peptides.

Aza-Amino Acid Surrogates

Compounds of Formula (I) to (XII) are stable at 37° C. in an aqueousmedium with a pH of about 7 (e.g., distilled water) for at least 30minutes, 60 minutes, 90 minutes, 1 hour, 2 hours, 3 hours, 4 hours or 5hours.

In certain embodiments, compounds of Formulas (I)-(XII) are protectedS-alkylthiocarbazates and could be used instead of amino acids andaza-amino acids in the syntheses of ureases, carbazides, semicarbazides,beta-peptides, azapeptides and other peptidomimetics and aza-peptideconjugates.

Various alkyl and the aromatic chains could be installed efficiently onthe unsubstituted S-alkylthiocarbazates with yields ranging between,e.g., about 50% and about 99%, about 55% and about 97%, about 60% andabout 95%, about 65% and about 95%, or about 70% to about 95%.

For example, protected unsubstituted S-alkylthiocarbazates may beprepared, e.g., by a reaction of a semi-protected hydrazine with achlorothioformate to form an unsubstituted alkylthio carbonyl hydrazine(unsubstituted S-alkylthiocarbazate). The reaction may be conducted at atemperature, e.g., of from about 20° C. to about 35° C. in, e.g.,4-dimethyl amino pyridine (DMAP), or a tertiary amine (e.g., DIPEA,Et3N, NMM, any Lutidine, apyridine, or collidine) for a time period,e.g., of from about 5 minutes to about 10 hours, from about 10 minutesto about 9 hours, from about 20 minutes to about 8 hours, from about 30minutes to about 6 hours, from about 45 minutes to about 5 hours, fromabout 1 hour to about 4 hours, or from about 1 hour to about 3 hours.

In certain embodiments, Mitsunobu reaction is used to prepare protectedunsubstituted S-alkylthiocarbazates. Mitsunobu reaction may be carriedout in ether solvents (e.g., tetrahydrofuran (THF)) where the substrate(thiocarbazate) is dissolved and treated with phosphine compounds likePh₃P, Dialkyl Azodicarboxylate (e.g., DEAD), in addition to appropriatealcohol.

In some embodiments, the protected unsubstituted ATCH (an unsubstitutedthiocarbazate) may be alkylated with an alcohol using the Mitsunobureaction to form a compound of Formula (I) to (XII). The reaction may beconducted at temperature of from about-5° C. to about 15° C. or fromabout 0° C. to about 10° C. in, e.g., THF, Dioxane, Et₂O, DCM, toluene,xylene, chlorobenzene, etc., for a time period of from about 5 minutesto about 8 hours, from about 10 minutes to about 6 hours, from about 20minutes to about 4 hours, from about 30 minutes to about 3 hours. Insome of these embodiments, 40% of Diethyl Azodicarboxylate (DEAD)solution in toluene, or Diisopropyl Azodicarboxylate, (DIAD), adiazopyridine, a diazoarene, or a Dialkyl Azo dicarboxylate may be addedto the reaction mixture drop-wise for a time period, e.g., of from about1 min to about 12 hours, about 1 min to about 11 hours, about 1 min toabout 10 hours, about 1 min to about 9 hours, about 1 min to about 8hours, about 1 min to about 7 hours, about 1 min to about 6 hours, about1 min to about 5 hours, about 1 min to about 4 hours, about 1 min toabout 3 hours, about 1 min to about 2 hours, or about 1 min to about 1hour. In some of these embodiments, a molar ratio of the protectedunsubstituted ATCH and the alcohol is from 0.5:1 to 1:1.5, and the molarratio of the protected unsubstituted ATCH and DEAD solution is fromabout 0.5:1.5 to about 1:3, and DEAD is added for a time period fromabout 1 min to about 1 hour. In some embodiments, from about 1 to about3 mmol of PPh₃ or another tri alkyl or tri aryl phosphine (e.g.,tricyclohexyl phosphine, tri butyl phosphine, and triphenyl phosphine)per mole of the protected unsubstituted ATCH (unsubstitutedthiosemicarbazides) is used.

Scheme 1 show examples of preparation of unsubstituted protected ATCHsfrom semi-protected hydrazines 1, 2, 3, or 4:

Protected unsubstituted ATCHs may have one available nitrogen foralkylation as in 5, or two nitrogen atoms as in 4 (Scheme 1).

Scheme 2 depicts examples of formation of substituted ACTHs byalkylation:

Alkylation may be, e.g., by the Mitsunobu reaction. Due to the differentreactivities of the two nitrogen atoms in 6, 7, or 8, the reaction willgrant an orthogonal functionalization of the nitrogen closer to thesulfur moiety, which is expected to be more nucleophilic than the othernitrogen under the Mitsunobu reaction due to the extra stability of theproduced conjugate base because of the sulfur atom effect (eq. 1 & eq. 2Scheme 2).

In scaffolds with a phthalimido-protecting group (e.g., 5), theMitsunobu alkylation is secured because only one NH is available foralkylation. The reaction could be performed, e.g., at 0° C. in THF as asolvent, where the alkylthio carbonyl hydrazine is mixed withstoichiometric or excess of alkyl or aryl phosphine like PPh₃, PCy₃,PBu₃ and appropriate alcohol with a careful drop-wise treatment ofDialkyl azodicarboxylate, e.g. Diethyl azodicarboxylate (DEAD) Reagent.The products may then be isolated by standard flash chromatography andcharacterized by mass and NMR Spectroscopies to afford the desired ACTH(substituted thiocarbazate) with moderate to excellent yields. Theyields for most of alcohols can be improved easily and efficientlyduring the R&D phase. Mintsunobu reaction conditions are mild andtolerated most of the alcohols needed to construct the ACTHs.

ATCHs may also be alkylated upon reflux with aldehydes. An example isdepicted in eq. 3 in scheme 2, where the reaction of scaffold 5 withparaformaldehyde in toluene under reflux conditions followed by standardacetylation produces the acetate 13 in a good yield.

Direct alkylation using NaH and alkyl halides may also be used tofunctionalize ACTHs, e.g., to produce cyclic derivatives like theAza-proline analog 14 depicted in eq. 4 in scheme 2.

Examples of substituted ACTHs that were prepared by the inventors aredepicted in Scheme 3 (along with some of the yields achieved):

The delicate indole moiety that is required to mimic the Tryptophaneside chain could, e.g., be introduced with no other side products in,e.g., 90% yield. Aza-aspartate surrogate 24 and Aza-Glutamate surrogate25 can be produced, e.g., with 93% and 53% yields respectively astert-butyl esters. The orthogonal choice of protecting groups is meantto expedite the elaboration of the synthetic options on all three sidesof the protected functionalities.

The amine-containing side chains of the aza-amino acid surrogates can bebuilt in a multistep fashion. For example, a simple and easilyaccessible starting materials like the 4-benzyl-1-butano can be used tosynthesize surrogate 27. A commercially available benzyl(4-hydroxybutyl) carbamate can be used, e.g., in excess (e.g., 2equivalents) to build the corresponding Aza-Lysine surrogate 29, e.g.,in 70% yield. Aza-arginine surrogate 30 could be constructed over twosteps with 35% using 1,3-propanediol as alkyl chain spacer. TheAza-Aspargine and the Aza-Glutamine surrogates can be accessed from thecorresponding Aza-Aspartate surrogate 24 and the Aza-Glutamate surrogate25, respectively. The Aza-Methionine surrogate 26 could be constructed,e.g., in 71% yield. Methionine surrogate 26 may be used as a buildingblock to make the corresponding semicarbazides. Aza-serine surrogate 13(Scheme 2) and the Aza-threonine surrogate may also be synthesized. Anacetylated Aza-serine surrogate 13 can be produced in a good yield,e.g., by treatment of 15 with paraformaldehyde under reflux conditionsin toluene followed by acetylation.

Protection groups other than phthalimidyl could also be used to preparecompounds of Formulas (I)-(XII). For example, carbamate-protectedscaffolds like 31 and 32 could be used for selective alkylation asdepicted in Scheme 4:

other examples for Boc protected alkyl thiocarbazates made by selectivefunctionalization using the Mitsuriobu reaction include, e.g.,

Carbamate-protected scaffolds like 31 and 32 could be built, e.g., fromthe commercially available t-butyl carbazate and the correspondingchlorothioformate, e.g., in 89% and 92% yields respectively. Bothcompounds 31 and 32 could be subjected separately to Mitsunobu reactionconditions and treated with stoichiometric amount of benzyl alcohol withslow addition of DEAD reagent. The alkylation favors the nitrogen atomcloser to the alkyl thiocarbonyl moiety and will give selectively theAzaphenylalanine surrogates 33 and 34, with, e.g., 70% and 85% yieldsrespectively. Template 32 could be used to rule out the steric hindranceeffect on the substitution. Compounds 33 and 34 could be isolated ascrystalline materials to confirm the substitution pattern by X-rayanalysis.

The substituted alkylthio carbonyl hydrazines (ATCHs), includingcompounds of Formulas (I) to (XII), could be used instead ofaza-amino-acids in the synthesis of ureases, carbazides, semicarbazides,beta-peptides, azapeptides, and other peptidomimetics and aza-peptideconjugates.

Activation of Acylthiols

In literature, the abstraction of the alpha C—H in thioethers by theeffect of N-chlorosuccinimide (NCS) is a well-documented reaction toactivate thioethers and use them as reactive synthons. Thioesters andthiocarboxylic acids also have been reported to react with NCS to givesulfonyl chlorides in good yields under harsh acidic conditions.Thiocarbamates have been reported to provide chloroformates when exposedto chlorine gas or refluxed with sulfuryl chloride SO₂Cl₂. Scheme 5Adepicts reactions of the literature:

The invention provides an alternative way of activating acylthiols(e.g., compounds of Formulas (I)-(XII)) by using halonium reagents(e.g., TCCA or a mixture of TCCA and TBACl) to convert the acylthiolsinto acylium intermediates in situ. Halonium reagents are expected toabstract an α-hydrogen to the sulfur atom and increase the migrationaptitude of the thiolate group to form an isocyanate intermediate in thecase of urea or carbonyl hydrazine or an acylium intermediate in thecase of thioester. Examplary reactions of the invention are depicted inScheme 5B:

The activation of acylthiols by using halonium reagents avoids, e.g., aneed for the unpractical and the harsh reaction conditions reported inthe literature. The activated acylthiols can then be coupled with anamine. The amine may be, e.g., an amino ester, an ester of an aminoacid, an amino ester of an aza-amino acid, a peptide, an aza-peptide, anamino acid, an aza-amino acid. If the amino ester, the ester of an aminoacid, the amino ester of the aza-amino acid, the peptide, theaza-peptide, the amino acid, or the aza-amino acid contains a groupselected from amino, amide, guanidino N, carboxyl, sulfhydryl, carboxyl,hydroxyl, indole, imidazole phenol, the group may be protected with aprotecting group selected from tert-butoxycarbonyl (Boc),9-fluorenylmethoxycarbonyl (Fmoc), or2-(3,5-dimethoxyphenyl)propan-2-yloxycarbonyl (Ddz), phthalimide (Phth),carboxybenzyl (Cbz), 2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl(Pbf), trityl or triphenylmethyl (Trt), t-butyl ester (OtBu), t-butylether (tBu), allyloxycarbonyl (Aloc), methoxytrimethylbenzene sulfonyl(Mtr), 4,4-dimethyloxybenzhydryl (Mbh),2,2,5,7,8-pentamethyl-chroman-6-sulfonyl chloride (Pmc),2,4,6-trimethoxybenzyl (Tmob), allyl ester (OAI), acetamidomethyl (Acm),and the like. In some embodiments, the amine is an amino ester (e.g., bet-butyl, p-methoxy benzyl ester, glycine ethyl ester, etc).

The common protecting groups appear to be stable under the conditionsnecessary to activate compounds of Formula (I)-(XII) by haloniumreagents. The reaction can be conducted, e.g., in chloroform,dichloromethane, acetone, acetonitrile, etc.

Thioureas and thiocarbonyl hydrazines have less reactive carbonylsystems toward nucleophilic addition, as compared to the correspondingthioesters due to the resonance effect of the neighboring nitrogen.Consequently, varying the reaction conditions could afford wide range offunctionalities.

Coupling of Activated Acylthiols

Once compounds of Formulas (I)-(XII) are activated they can be coupledto an amine to make unsubstituted and substituted semicarbazides, e.g.,azapeptides and other peptidomimetics and aza-amino acid conjugates. Theamine may be, e.g., an amino ester, an ester of an amino acid, an aminoester of an aza-amino acid, a peptide, an aza-peptide, an amino acid, anaza-amino acid. If the amino ester, the ester of an amino acid, theamino ester of the aza-amino acid, the peptide, the aza-peptide, theamino acid, or the aza-amino acid contains a group selected from amino,amide, guanidino N, carboxyl, sulfhydryl, carboxyl, hydroxyl, indole,imidazole phenol, the group may be protected with a protecting groupselected from tert-butoxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl(Fmoc), or 2-(3,5-dimethoxyphenyl)propan-2-yloxycarbonyl (Ddz),phthalimide (Phth), carboxybenzyl (Cbz),2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl (Pbf), trityl ortriphenylmethyl (Trt), t-butyl ester (OtBu), t-butyl ether (tBu),allyloxycarbonyl (Aloe), methoxytrimethylbenzene sulfonyl (Mtr),4,4-dimethyloxybenzhydryl (Mbh),2,2,5,7,8-pentamethyl-chroman-6-sulfonyl chloride (Pmc),2,4,6-trimethoxybenzyl (Tmob), allyl ester (OAI), acetamidomethyl (Acm),and the like. In some embodiments, the amine is an amino ester (e.g., bet-butyl, p-methoxy benzyl ester, glycine ethyl ester, etc).

In certain embodiments, A of compounds of Formulas (I)-(XII) isphthalimidyl, and these compounds are activated by TCCA to convert theminto acylium intermediates in situ by an exemplary reaction depicted inScheme 6, and the resulting acylium intermediate is reacted with anamine (e.g., glycine ethyl ester), e.g., in DIPEA to form a substitutedor unsubstituted semicarbazide:

In certain embodiments, the activated acylthiol is coupled with an aminoester. The careful and orthogonal choice of amino acid ester isimportant. Acid labile esters (e.g., like t-butyl, p-methoxy benzylesters, etc.) are compatible with the phthalimide group. However,careful handling of the cleaving of the phthalimido group undercontrolled temperature is also feasible. The use of amino esters ascoupling partners reduces unwanted side products formed as a result ofreactions of free carboxylic acids with, e.g., TCCA, a mixture of TCCAand TBACl, etc.

In certain embodiments, A of compounds of Formulas (I)-(XII) is Boc, andthese compounds are activated by halonium reagents to convert them intoacylium intermediates in situ by a reaction depicted in Scheme 8, andthe resulting acylium intermediate is then reacted with an amine (e.g.,an aminoacid ester) to form an unsubstituted or substitutedsemicarbazide:

The unsubstituted and substituted semicarbazides can be used, e.g., asdiazide surrogates, to make azapeptides and other peptidomimetics andaza-amino acid conjugates, e.g., in solid phase peptide synthesis (SPPS)or a solution peptide synthesis.

Synthesis of Ureases, Carbazides, Semicarbazides, Beta-Peptides,Azapeptides and Other Peptidomimetics and Aza-Amino Acid Conjugates

Compounds of Formulas (I) to (XII) can be coupled in a linear, stepwise,chain-lengthening fashion to each other, amino acids, aza-amino acids,peptides, azapeptides, and azatides by solution phase, solid phase andmixed solution/solid phase synthetic methodologies to constructsemicarbazides, beta-peptides, azapeptides and other peptidomimetics andaza-amino acid conjugates. For example, when the synthons are used inthe synthesis of an azapeptide, the azapeptide is preferably produced ina yields ranging between about 55% and 99% (by weight) (e.g., from about60% to about 95% or from about 65% to about 95%). The yield may, e.g.,be about 55%, about 60%, about 65%, about 75%, about 80%, about 85%,about 90%, about 95%, about 97%, or about 99%. In certain embodiments,the average yield after both activating and coupling is greater than 85%for most of the amino acid residues combinations.

Compounds of Formula (I) to (XII) can also be used, e.g., assub-monomers to elongate and/or cap peptides and azapeptides.

For example, in certain embodiments, compounds of Formula (I) to (XII)may be activated by a halonium reagent(s) (e.g., TCCA or a mixture ofTCCA and TBACl), and the activated compound may be coupled, e.g., aprotected or unprotected aza-amino acid; a protected or unprotected apeptide; a protected or unprotected azapeptide; a protected orunprotected azatide; or a protected or unprotected compound of Formula(I) to (XII); or a protected or unprotected hydrazine, by eithersolution or solid phase synthetic methodologies, e.g., to form anaza-peptide. The amino acid, the aza-amino acid, the peptide, theazapeptide, compound of Formula (I) to (XII) may each be unsubstitutedor substituted with one or more of the following: a halogen (Cl, F, orBr), a C₁-C₆ alkyl (e.g., methyl), hydroxyl, methoxyl, ethoxyl,propoxyl, a C₁-C₆ haloalkyl (e.g., a chloromethyl, a fluromethyl, etc.).

The methods of the invention may be used to synthesize, e.g.,azapeptides from 2 to 200 mers in length, e.g., di-azatides,tri-azatides, tetra-azapeptides, penta-azapeptides, etc.

In certain embodiments, the method of preparing an azapeptide or anazatide comprises hydrolysing a peptide into fragments and reacting oneor more fragments with a compound of Formula (I) to (XII).

In certain embodiments, the method of preparing an azapeptide or anazatide comprises cleaving a peptide into fragments and reacting one ormore fragments with a compound of Formula (I) to (XII).

In certain embodiments, the method of preparing an azapeptide or anazatide comprises cleaving an end of a peptide, and reacting the cleavedpeptide with a compound of Formula (I) to (XII).

In certain embodiments, the method of preparing an azapeptide or anazatide comprises reacting a compound of Formula (I) to (XII) with atruncated peptide.

In certain embodiments, a method of azapeptide or azatide synthesiscomprise reacting (i) a compound of formula (I) to (XII) with (ii) apeptide to form the azapeptide or azatide, wherein the azapeptide orazatide is a compound of formula (XVI).

In certain embodiments, a compound of formula (I) to (XII) is asubstituted semicarbazide. The substituted semicarbazide may beprepared, e.g., by activating a compound of formula (I) to (XII) with ahalonium reagent(s) (e.g., TCCA or a mixture of TCCA and TBACl). Theactivated compound is then reacted with an amine, e.g., in the presenceof diisopropylethylamine (DIPEA, Et3N, Me3N, NMO, Lutidines, pyridines,DMAP, collidines, or any tertiary amine). In some of these embodiments,the compound of formula (I) to (XII), TCCA and TBACl are used in a molarratio of about 1:1:1. The molar ratio of the compound of formula (I) to(XII) to the amine may, e.g., be from about 0.5:1 to 1:1.5. In someembodiments, DCM is added to the reaction mixture. The reaction may beconducted for a time period of from about 5 minutes to about 5 hours ata temperature of from about −5° C. to about 30° C. In some embodiments,the reaction mixture is cooled before addition of the amine. In someembodiments, the reaction mixture may be treated with saturated solutionof sodium thiosulfate.

In certain embodiments, a compound of formula (I) to (XII) is asubstituted semicarbazide. The substituted semicarbazide may beprepared, e.g., by exposing a compound of formula (I) to (XII) with TCCAor a mixture of TCCA and TBACl to activate the compound, and thenreacting the activated compound with an amine, e.g., in the presence ofdiisopropylethylamine (DIPEA, Et3N, Me3N, NMO, Lutidines, pyridines,DMAP, collidines, or a tertiary amine). In some of these embodiments,the compound of formula (I) to (XII), TCCA and TBACl are used in a molarratio of about 1:1:1. The molar ratio of the compound of formula (I) to(XII) to the amine may, e.g., be from about 0.5:1 to 1:1.5. In someembodiments, DCM is added to the reaction mixture. The reaction may beconducted for a time period of from about 5 minutes to about 5 hours ata temperature of from about −5° C. to about 30° C. In some embodiments,the reaction mixture is cooled before addition of the amine. In someembodiments, the reaction mixture may be treated with saturated solutionof sodium thiosulfate.

Synthesis of Compounds of Formula (XVI)

In certain embodiments, compounds of Formulas (I) to (XV) are used tosynthesize compounds of Formula (XVI). Compound of Formula (XVI) may beused in drug discovery, diagnosis, prevention, inhibition, and treatmentof diseases. For example, compound of Formula (XVI) may be used toinhibit one or more symptom(s) of the diseases. Compound of Formula(XVI) may be used to completely alleviate one or more symptom(s) of thediseases. In certain embodiments, compounds of Formula (XVI) areazapeptide analogues of therapeutic and diagnostic peptides and are moreresistant to hydrolysis and/or enzymatic degradation than thetherapeutic and diagnostic peptides.

Compound of Formula (XVI) may comprise from 2 to 200 carbonyl group(s).For example, compound of Formula (XVI) may comprise 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 36, 37, 38, 39, 40, 41, 43, 44, 56, or 166carbonyl groups. In certain embodiments, compound of Formula (XVI)comprises from 2 to 60 carbonyl groups, from 2 to 50 carbonyl groups,from 2 to 40 carbonyl groups, from 2 to 30 carbonyl groups, from 2 to 25carbonyl groups, from 2 to 20 carbonyl groups, from 2 to 15 carbonylgroups, from 2 to 12 carbonyl groups, from 2 to 10 carbonyl groups, from2 to 9 carbonyl groups, from 3 to 40 carbonyl groups, from 3 to 30carbonyl groups, from 3 to 25 carbonyl groups, from 3 to 20 carbonylgroups, from 3 to 15 carbonyl groups, from 3 to 12 carbonyl groups, from3 to 10 carbonyl groups, or from 3 to 9 carbonyl groups.

In certain embodiments, compound of Formula (XVI) comprises from 2 to200 carbonyl groups and at least one α-nitrogen covalently bound to atleast one of said carbonyl groups, and have a greater bioavailability(e.g., oral, transdermal, and/or intranasal) than a compounds that lacksthe at least one α-nitrogen. In certain embodiments, the α-nitrogen isnot at the N-termini and not at the C-termini of the compounds ofFormula (XVI), rather it is at a cleavage or hydrolysis site(s) of thepeptide.

In certain embodiments, compounds of Formula (XVI) comprises a backbonecomprising from 2 to 200 carbonyl groups and α-nitrogen covalently boundto at least one of said carbonyl groups, and is therapeuticallyeffective for the treatment of a disorder in a subject. Compounds ofFormula (XVI) may, e.g., inhibit or completely alleviate one or moresymptom(s) of the disorder in the subject.

In certain embodiments, the compound of Formula (XVI) comprises from 2to 60 carbonyl groups.

In certain embodiments, the compound of Formula (XVI) is linear.

In certain embodiments, the compound of Formula (XVI) is cyclic.

In certain embodiments, the compound of Formula (XVI) is pegylated.

In certain embodiments, the compound of Formula (XVI) is conjugated toan immunoglobulin.

In certain embodiments, the compound of Formula (XVI) comprisesα-nitrogen at the N-terminus of its backbone.

In certain embodiments, the compound of Formula (XVI) comprisesα-nitrogen at the C-terminus of its backbone.

In certain embodiments, the compound of Formula (XVI) comprises twocarbonyl groups and two α-nitrogens.

In certain embodiments, the compound of Formula (XVI) comprises threecarbonyl groups and one α-nitrogen.

In certain embodiments, the compound of Formula (XVI) comprises threecarbonyl groups and two α-nitrogens.

In certain embodiments, the compound of Formula (XVI) comprises threecarbonyl groups and three α-nitrogens.

In certain embodiments, the compound of Formula (XVI) comprises fourcarbonyl groups and one α-nitrogen.

In certain embodiments, the compound of Formula (XVI) comprises fourcarbonyl groups and two α-nitrogens.

In certain embodiments, the compound of Formula (XVI) comprises fourcarbonyl groups and three α-nitrogens.

In certain embodiments, the compound of Formula (XVI) comprises fourcarbonyl groups and four α-nitrogens.

In certain embodiments, compounds of Formula (I) to (XII) are used toprepare aza-analogues of A-6, A-623 (AMG-623), A-71378, A-75998,Abarelix (PPI-149), ABT-510, AC-100, AC-162352 (PYY 3-36), AC-253,AC-2592, AC-625, ACV-1, ADH-1, AEZS-108 (AN-152) (ZEN-008), AF-37702,Afamelanotide (EP-1647) (CUV-1647) (Melanotan I), AG2/102, AG-284,AI-502, AKL-0707 (LAB GHRH), Albiglutide (GSK-716155), Albuvirtide,ALG-889, Alloferon, Allotrap 2702 (B-2702), ALTY-0601, ALX-40-4C,Ambamustine (PTT-119), Anaritide, Antagonist G (PTL-68001), AOD-9604,APL-180, ATN-161, Atosiban (ORF-22164), Atriopeptin, Aviptadil(PSD-510), Avorelin (EP-23904), AZD-2315, Azetirelin (YM-14673),AZX-100, B27PD, BA-058, Barusiban (FE-200400), BAY-73-7977, BDM-E,BGC-728, BIM-23190, BIM-44002, BIO-1211, Bivalirudin (BG-8865),BMS-686117, Bremelanotide (PT-141), BRX-0585, Buserelin, Calcitonin(Human), Calcitonin (Salmon), Carbetocin, Carfilzomib (PR-171),Cargutocin (Y-5350), Carperitide (SUN-4936), Casokefamide, CB-182804,CB-183315, CBP-501, CBT-101, CCK (25-33), CD-NP, Cemadotin (LU-103793),Cetrorelix (NS-75), CG-77X56, CGRP (LAB-CGRP), Chlorotoxin (TM-601),Cilengitide (EMD-121974) (EMD-85189), CJC-1008 (DAC: Dynorphin A),CJC-1131 (DAC:GLP-1), CJC-1134 (PC-DAC) (Exendin-4), CJC-1295 (DAC:GRF),Cnsnqic-Cyclic (802-2), Compstatin (POT-4), Conantokin G, Contulakin G(CGX-1007), Corticorelin (NEU-3002), CP-95253, C-peptide (SPM-933),CR-665, CR-845, CTCE-0214, CTCE-9908, CTS-21166 (ASP-1702) (ATG-Z1)(OM-00-3) (OM-99-2), CVX-045, CVX-060, CVX-096 (PF-4856883), CZEN-002,D-4F (APP-018), Danegaptide (ZP-1609) (WAY-261134) (GAP-134),Davalintide (AC-2307), Davunetide (AL-108) (AL-208), Degarelix (FE200486), Delmitide (RDP-58), Deltibant (CP-0127), Deslorelin,Desmopressin, Detirelix (RS-68439), DG-3173 (PTR-3173), Didemnin B(NSC-325319), Dirucotide (MBP-8298) Disitertide (NAFB-001) (P-144),DMP-728 (DU-728), dnaJP1 (AT-001), Dopastatin (BIM-23A760), DPK-060,DRF-7295, DSC-127, Dynorphin A, E-2078, EA-230, Ebiratide (Hoe-427),Edotreotide (SMT-487), Edratide (TV-4710), Efegatran (LY-294468),Elcatonin, Eledoisin (ELD-950), Elisidepsin (PM-02734), EMD-73495,Enfuvirtide (T-20), EP-100, EP-51216 (EP-51389), Eptifibatide (C68-22),ET-642 (RLT-peptide), ETRX 101, Examorelin (EP-23905) (MF-6003),Exenatide (AC-2993) (LY-2148568), Exsulin (INGAP Peptide), F-991,FAR-404, FE 202158, Felypressin, FGLL, Frakefamide (LEF-576) (SPD-759)(BCH-3963), FX-06, Ganirelix (Org-37462) (RS-26306), Glaspimod(SKF-107647), Glatiramer (COP-1), Glucagon, Glucosamyl muramyltripeptide, Glutoxim (NOV-002), Glypromate, GMDP, Golotimod (SCV-07),Goralatide (BIM-32001), Goserelin (ICI-118630), GPG-NH₂, GTP-200,GTP-300, H-142, Hemoparatide (PTH (1-37)), Hexapeptide copper II(PC-1358), Histrelin, hLF (1-11), HP-228, 1-040302 (KUR-112), Icatibant(JE-049) (HOE-140), lcrocaptide (ITF-1697), IMX-942, lpamorelin(NNC-26-0161), IPP-201101, Iseganan (IB-367), ISF402, Iturelix(ORF-23541), JTP-2942, KAI-1455, KAI-1678, KM-9803, KP-101 (GIMP-1),L-346670, L-364343, Labradimil (RMP-7), Lagatide (BN-52080), Lanreotide(ITM-014), Larazotide (AT-1001) (SPD-550), Leconotide (AM-336),Leuprolide (SOT-375), Linaclotide (MD-1100) (MM-41775), Liraglutide(NN-2211), Lixisenatide (AVE-0010) (ZP-10), LSI-518P, Lucinactant,Lusupultide (BY-2001), LY-2189265, LY-2510924, LY-548806, LYN-001,Lyprssin, MER-104, Met-enkephalin (INNO-105), Metkephamide (LY-127623),Mifamurtide (CGP-19835) (MLV-19835), Montirelin (CG-3703), MPL-TLB100,MS peptide, MT-11 (PT-14), Murabutide (VA-101) (CY-220), Muramyltripeptide, Nafarelin (RS-94991), NBI-6024, Nemifitide (INN-00835),Neogen, Nepadutant (MEN-11420), Nesiritide, Nifalatide (BW942C),NNZ-2566, NP-213, NFC-567, NPY (24-36) (PTL-041120), NT-13, Obinepitide(TM-30338), Octreotide (SMS-201-995), Oglufanide (IM-862), OGP 10-14L,Omiganan (CPI-226), OP-145, ORG-2766 Org-42982 (AG-4263), Ornithinevasopressin, Oxytocin, Ozarelix (D-63153) (SPI-153), p-1025, P-113(PAC-113), Pasireotide (SOM-230), peg-TPOmp (RWJ-800088), Pentigetide(TA-521), Pep-F (5K), Peptide renin inhibitor, Peptide T (AIDS000530),Peptide YY 3-36, Pexiganan (MSI-78), PF-4603629, PI-0824, PI-2301,PL-3994, PLD-116, PMX-53, POL-6326, Posatirelin, PPI-1019, Pralmorelin,Pramlintide, Protirelin, PTH (7-34), PTHrP-(1-36), PTL-0901, PXL-01,R-1516, R-15-K, R-7089, RA peptide, Ramorelix (Hoe-013), RC-3095,Re-188-P-2045 (P2045), rGRF, Romiplostim (AMG-531), Romurtide (DJ-7041),ROSE-010 (GTP-010) (LY-307161), Rotigaptide (ZP-123) (GAP-486),Rusalatide (TP-508), SAN-134, Saralasin (P-113), Secretin (human)(PGN-52) (R-52), Secretin (human) (RG-1068), Semaglutide (NN-9535),SGS-111, Sifuvirtide, SKF-101926, SKF-105494, SKF-110679 (U-75799E),Soblidotin (YHI-501) (TZT-1027), Somatostatin, Somatostatin (D-Trp,D-Cys analog), SP-304 (Guanilib), SPC-3, SPI-1620, SST analog,SUN-11031, SUN-E7001 (CS-872), SYN-1002, Tabilautide (RP-56142),TAK-448, TAK-683, Taltirelin (TA-0910), Tasidotin (ILX-651)(BSF-223651), Taspoglutide (BIM-51077), TCMP-80, Teduglutide (ALX-0600),Teriparatide (LY-333334), Terlakiren (CP-80794), Terlipressin,Tesamorelin (TH-9507), Teverelix (EP-24332), TH-0318, TH-9506,Thymalfasin, Thymodepressin, Thymonoctan (FCE-25388), Thymopentin(TP-5), Thymosin beta-4, Tifuvirtide (R-724) (T-1249), Tigapotide(PCK-3145), Tiplimotide (NBI-5788), TKS-1225 (Oxyntomodulin), Tth-232(CAP-232)(TT-232), TM-30339, TP-9201, TRI-1144, Tridecactide (AP-214),Triletide (Z-420) (ZAMI-420), Triptorelin (WY-42462), TT-223, (E1-INT),TT-235, TX14(A), Tyroserleutide (CMS-024), Tyroservatide (CMS-024-02),Ularitide (CDD-95-126) (ESP-305), Unacylated ghrelin (AZP-01) (TH-0332),Urocortin 11, Vapreotide (RC-160), Vasopressin, VIR-576, Xen-2174,XG-102, XOMA-629, Ziconotide (SNX-111), ZP-120, or ZP-1846.

In certain embodiments, compounds of Formula (I) to (XII) are used toprepare aza-analogues of AC-2592, AC-625, Anaritide, APL-180,Atriopeptin, BGC-728, Carperitide (SUN-4936), CD-NP, CG-77X56, D-4F(APP-018), Danegaptide (ZP-1609) (WAY-261134) (GAP-134), DMP-728(DU-728), Efegatran (LY-294468), EMD-73495, Eptifibatide (C68-22),ET-642 (RLT-peptide), FE 202158, FX-06, Icatibant (JE-049) (HOE-140),lcrocaptide (ITF-1697), KAI-1455, KM-9803, L-346670, L-364343, LSI-518P,Nesiritide, Peptide renin inhibitor, PL-3994, Rotigaptide (ZP-123)(GAP-486), Saralasiri (P-113), SKF-105494, Terlakiren (CP-80794),Tridecactide (AP-214), Ularitide (CDD-95-126) (ESP-305), Urocortin 11,Ziconotide (SNX-111), or ZP-120; and have utility in the treatment ofcardiovascular diseases (e.g., alleviate one or more symptom(s) of thecardiovascular disease).

In certain embodiments, compounds of compounds of Formula (I) to (XII)are used to prepare aza-analogues of Azetirelin (YM-14673), ConantokinG, Corticorelin (NEU-3002), CTS-21166 (ASP-1702) (ATG-Z1) (OM-00-3)(OM-99-2), Davunetide (AL-108) (AL-208), Deltibant (CP-0127), Ebiratide(Hoe-427), FGLL, Glypromate, JTP-2942, Montirelin (CG-3703), Nemifitide(INN-00835), NNZ-2566, NT-13, ORG-2766, Peptide T (AIDS000530),Posatirelin, PPI-1019, Protirelin, Secretin (human) (RG-1068), SGS-111,Taltirelin (TA-0910), XG-102, or Ziconotide (SNX-111), and have utilityin the treatment of CNS disorders (e.g., alleviate one or moresymptom(s) of the CNS disorder):

In certain embodiments, compounds of Formula (I) to (XII) are used toprepare aza-analogues of A-6, Abarelix (PPI-149), ABT-510, ADH-1,AEZS-108 (AN-152) (ZEN-008), Ambamustine (PTT-119), Antagonist G(PTL-68001), ATN-161, Avorelin (EP-23904), Buserelin, Carfilzomib(PR-171), CBP-501, Cemadotin (LU-103793), Chlorotoxin (TM-601),Cilengitide (EMD-121974) (EMD-85189), CTCE-9908, CVX-045, CVX-060,Degarelix (FE 200486), Didemnin B (NSC-325319), DRF-7295, Edotreotide(SMT-487), Elisidepsin (PM-02734), EP-100, Glutoxim (NOV-002),Goralatide (BIM-32001), Goserelin (ICI-118630), Histrelin, Labradimil(RMP-7), Leuprolide (SOT-375), LY-2510924, Met-enkephalin (INNO-105),Mifamurtide (CGP-19835) (MLV-19835), Muramyl tripeptide, Ozarelix(D-63153) (SPI-153), POL-6326, Ramorelix (Hoe-013), RC-3095,Re-188-P-2045 (P2045), Romurtide (DJ-7041), Soblidotin (YHI-501)(TZT-1027), SPI-1620, Tabilautide (RP-56142), TAK-448, TAK-683,Tasidotin (ILX-651) (BSF-223651), Teverelix (EP-24332), Tigapotide(PCK-3145), TLN-232 (CAP-232)(TT-232), Triptorelin (WY-42462),Tyroserleutide (CMS-024), Tyroservatide (CMS-024-02), ZP-1848, inZT0131; and have utility in the treatment of oncological conditions(e.g., alleviate one or more symptom(s) of the an oncologicalcondition).

In certain embodiments, compounds of Formula (I) to (XII) are used toprepare aza-analogues of A-623 (AMG-623), AG-284; AI-502, Allotrap 2702(B-2702), AZD-2315, Cnsnqic-Cyclic (802-2), Delmitide (RDP-58),Dirucotide (MBP-8298) Disitertide (NAFB-001) (P-144), dnaJP1 (AT-001),Edratide (TV-4710), F-991, FAR-404, Glaspimod (SKF-107647), Glatiramer(COP-1), GMDP, IPP-201101, Icatibant (JE 049)(HOE-140), MS peptide,Org-42982 (AG-4263), Pentigetide (TA-521), PI-0824, PI-2301, PLD-116,PMX-53, PTL-0901, RA peptide, TCMP-80, Thymodepressin, Thymopentin(TP-5), Tiplimotide (NBI-5788), or ZP-1848; and have utility in thetreatment of allergy and immunology disorders.

In certain embodiments, compounds of Formula (I) to (XII) are used toprepare aza-analogues of A-71378, AC-162352 (PYY 3-36), AC-253, AG2/102,AKL-0707 (LAB GHRH), Albiglutide (GSK-716155), AOD-9604, BAY-73-7977,BIM-44002, BMS-686117, BRX-0585, CJC-1131 (DAC:GLP-1), CJC-1134 (PC-DAC)(Exendin-4), CJC-1295 (DAC:GRF), CP-95253, CVX-096 (PF-4856883),Davalintide (AC-2307), Exenatide (AC-2993) (LY-2148568), Exsulin (INGAPPeptide), Glucagon, ISF402, Liraglutide (NN-2211), Lixisenatide(AVE-0010) (ZP-10), LY-2189265, LY-548806, nafarelin (RS 94991),NBI-6024, Obinepitide (TM-30338), Peptide YY 3-36, PF-4603629,Pramlintide, R-7089, Semaglutide (NN-9535), SST analog, SUN-E7001(CS-872), Taspoglutide (BIM-51077), Tesamorelin (TH-9507), TH-0318,TKS-1225 (Oxyntomodulin), TM-30339, TT-223 (E1-INT), Unacylated ghrelin(AZP-01) (TH-0332), or ZT0131, and have utility in the treatment ofmetabolic disorders (e.g., alleviate one or more symptom(s) of ametabolic disorder).

In certain embodiments, compounds of Formula (I) to (XII) are used toprepare aza-analogues of A-75998, Buserelin, Cetrorelix (NS-75),Detirelix (RS-68439), Ganirelix (Org-37462) (RS-26306), Iturelix,Nafarelin (RS-94991), or triproletin (WY-42462); and have utility in thetreatment of fertility.

In certain embodiments, compounds of Formula (I) to (XII) are used toprepare aza-analogues of AC-100 and p-1025, and have utility in thetreatment of dental disorders.

In certain embodiments, compounds of Formula (I) to (XII) are used toprepare aza-analogues of ACV-1, Conantokin G, CJC-1008 (DAC: DynorphinA), Contulakin G, (CGX-1007), CR-665, CR-845, Dynorphin A, E-2078,Felypressin, Frakefamide (LEF-576) (SPD-759) (BCH-3963), HP-228,Icatibant (JE-049) (HOE-140), KAI-1678, Leconotide (AM-336),Metkephamide (LY-127623), MPL-TLB100, NT-13, SYN-1002, TX14(A),Xen-2174, and Ziconotide (SNX-111); and have utility in the treatment ofpain.

In certain embodiments, compounds of Formula (I) to (XII) are used toprepare aza-analogues of Afamelanotide (EP-1647) (CUV-1647) (MelanotanI), AZX-100, DPK-060, DSC-127, Hemoparatide (PTH (1-37)), Hexapeptidecopper H (PC-1358), Pexiganan (MSI-78), PTH (7-34), PXL-01, SKF-110679(U-75799E), or Thymosin beta-4; and have utility in the treatment ofdermatologic conditions (e.g., alleviate one or more symptom(s) of adermatological condition).

In certain embodiments, compounds of Formula (I) to (XII) are used toprepare aza-analogues of AF-37702, Bivalirudin (BG-8865), carfilzomib,(PR-171), CTCE-0214, ETRX 101, H-142, OGP 10-14L, Ornithine vasopressin,peg-TPOmp (RWJ-800088), R-1516, Romiplostim (AMG-531), and TP-9201; andhave utility in the treatment of hematology disorders (e.g., alleviateone or more symptom(s) of a hematological disorder).

In certain embodiments, compounds of Formula (I) to (XII) are used toprepare aza-analogues of Albuvirtide, ALG-889, Alloferon, ALX-40-4C,CB-182804, CB-183315, CZEN-002, Enfuvirtide (T-20), Glucosamyl muramyltripeptide, Golotimod (SCV-07), GPG-NH2, hLF (1-11), IMX-942, Iseganan(IB-367), Murabutide (VA-101) (CY-220), Neogen, NP-213, Oglufanide(IM-862), Omiganan (CPI-226), OP-145, p-1025, P-113 (PAC-113), Pep-F(5K), R-15-K, Sifuvirtide, SPC-3, Thymalfasin, Thymonoctan (FCE-25388),Tifuvirtide (R-724) (T-1249), TRI-1144, VIR-576, or XOMA-629; and haveutility as an antimicrobial or antiviral agent.

In certain embodiments, compounds of Formula (I) to (XII) are used toprepare aza-analogues of ALTY-0601, B27PD, BDM-E, BIM-23190, CBT-101,Compstatin (POT-4), Eledoisin (ELD-950), and LYN-001, and have utilityin the treatment of ophthalmologic disorders (e.g., alleviate one ormore symptom(s) of an ophthalmologic disorder).

In certain embodiments, compounds of Formula (I) to (XII) are used toprepare aza-analogues of Atosiban (ORF-22164), Barusiban (FE-200400),Carbetocin, Cargutocin (Y-5350), Deslorelin, Oxytocin, or TT-235, andhave utility in the treatment of OB-GYN disorders.

In certain embodiments, compounds of Formula (I) to (XII) are used toprepare aza-analogues of Aviptadil (PSD-510), Bremelanotide (PT-141),C-peptide (SPM-933), Desmopressin, EA-230, Lypressin, MER-104, MT-11(PT-14), SKF-101926, or Vasopressin, and have utility in the treatmentof urologic conditions (e.g., alleviate one or more symptom(s) of aurologic condition).

In certain embodiments, compounds of Formula (I) to (XII) are used toprepare aza-analogues of AC-100, BA-058, Calcitonin (Human), Calcitonin(Salmon), Elcatonin, I-040302 (KUR-112), PTHrP-(1-36), Rusalatide(TP-508), SAN-134, Teriparatide (LY-333334), or ZT031; and have utilityin the treatment of bones and connective tissue disorders.

In certain embodiments, compounds of Formula (I) to (XII) are used toprepare aza-analogues of BIO-1211, CGRP (LAB-CGRP), Glucosamyl muramyltripeptide, Icrocaptide (ITF-1697), Lucinactant, Lusupultide (BY-2001),NPC-567, NPY (24-36) (PTL-041120), or Secretin (human) (PGN-52) (R-52);and have utility in the treatment of respiratory conditions (e.g.,alleviate one or more symptom(s) of a respiratory condition).

In certain embodiments, compounds of Formula (I) to (XII) are used toprepare aza-analogues of Casokefamide, CCK (25-33), Lagatide (BN-52080),Larazotide (AT-1001) (SPD-550), Linaclotide (MD-1100) (MM-41775),Nepadutant (MEN-11420), Nifalatide (BW942C), ROSE-010 (GTP-010)(LY-307161), Somatostatin, Somatostatin (D-Trp, D-Cys analog), SP-304(Guanilib), Teduglutide (ALX-0600), Terlipressin, Triletide (Z-420)(ZAMI-420), Vapreotide (RC-160), ZP-1846, or ZP-1846; and have utilityin the treatment of gastroenterologic disorders (e.g., alleviate one ormore symptom(s) of a gastroenterologic disorder).

In certain embodiments, compounds of Formula (I) to (XII) are used toprepare aza-analogues of CJC-1295 (DAC:GRF), DG-3173 (PTR-3173),Dopastatin (31M-23A760), EP-51216 (EP-51389), Examorelin (EP-23905)(MF-6003), GTP-200 (GTP-300), lpamorelin (NNC-26-0161), Iturelix(ORF-23541), KP-101 (GHRP-1), Lanreotide (ITM-014), Octreotide(SMS-201-995), Pasireotide (SOM-230), Pralmorelin, rGRF, SUN-11031,TH-9506, ZT0131, or vapreotide (RC-160); and have utility in thetreatment of endocrinology disorders (e.g., alleviate one or moresymptom(s) of an endocrinology disorder).

Example 1 Activation of the Alkylthio Hydrazine Scaffolds

Template 21 was reacted with one equivalent of TCCA in dichloromethane(DCM) as a solvent, the reaction took place at room temperature and wasmonitored by TLC, we barely observed a change in R_(f) values compare tothe starting material on TLC (Scheme 6). Yet, the mass spectrometryanalysis indicated the disappearance of the starting material.

Next, 2.0 equivalents of protected amino acid like glycine ethyl esteraccompanied with four equivalents of diisopropylethylamine (DIPEA) wasadded. The corresponding substituted semicarbize 36 was observed as theonly product on TLC. Later, the product was isolated with 94% yield(Scheme 6).

The same experiment was repeated without TCCA as a control experiment.Only the starting material was recovered, without any evidence ofsubstitution at the thiocarbonyl moiety. When the control experiment wasrepeated with heat at 60° C., the amine attack favors the carbonylmoiety at the phthalimido-protecting group.

Next, the compatibility of the reaction conditions with differentsolvent systems was tested. The reaction performed well in chloroform,dichloromethane, acetone, and acetonitrile. We noticed that the reactionproceeded faster in acetonitrile and showed vigorous behavior indimethylformamide (DMF), but gave an unclean profile on TLC. Tounderstand the reaction mechanism, we opted to isolate the intermediateafter the TCCA addition.

The reaction was repeated with two equivalents of TCCA to securecomplete conversion of the starting material especially bothintermediate, and the starting material showed the same R_(f) valuesunder different mobile phases. After evaporating the volatile solvent,the intermediate was purified using standard chromatography without theneed to do any work-up. After purification, we collected needle-shapedcrystals; the X-ray analysis proved the formation of hydrazine carbonylchloride (chloroformate) intermediate. Surprisingly, this intermediatewas stable under standard purification protocol (Scheme 6)

The scope of this reaction was then studies. First, a template havingphthalimido protecting group was decorated with different side chains.The results are summarized in (Scheme 7):

After optimizations, the reaction proceeded with one equivalent of TCCAand 1.5 equivalent of amine for coupling. The reaction tolerated most ofthe alkyl and aromatic side chains with no evidence of formation ofchlorinated side products or any other side products.

One of the possible side products in this reaction is the formation ofHCl gas or in solution, as a result, the treatment of ATCHs with TCCA inthe first step is expected to be slightly acidic, to test the durabilityof acid reactive compounds with this protocol we chose acid labilemoieties like the t-butyl esters 24 and 25. The correspondingsemicarbazides 49 and 50 were isolated in good yields with no hydrolyzedesters as side products.

The side chains with amino group moieties might be reactive to the TCCAreagent. However, when the reaction performed on 29 and 30, thecorresponding semicarbazides 41 and 48 respectively were collected inlow to moderate yields. This might be because of the sensitivity of theBoc group and because of the reactivity of the NH carbamates to thechloronium species.

This protocol is proved to be also useful to prepare tripeptidesegments. Compound 39 is a good example, which was made from ATCH 21 andprotected serine-proline dipeptide.

To extend the scope of this reaction to cover more labile and delicatescaffolds like the Boc protected moieties, we determined to optimize thereaction condition by tuning the TCCA reactivity. Inventors found thatthe addition of one equivalent of tetrabutylammonium chloride reagent iscapable of increasing the reaction rate; as a result, running thereaction at a lower temperature is conceivable. Inventors explored thenew reaction conditions on the previous examples, and confirmed enhancedperformance and higher yields for most of these depicted examples inscheme 7.

Encouraged with these findings, inventors tested the new class of Bocprotected thiocarbazates 14 and 33. This reaction preceded smoothly withgood to high yields (Scheme 8): Scaffolds 14 and 33 were treatedseparately with one equivalent of TCCA and one equivalent TBACl at 0°C., then was treated with valine t-butyl ester to give the correspondingsemicarbazides with 68% and 89% yields respectively.

According to the literature, 51 can be made over four steps throughreductive-amination and coupling using phosgene as the acylating agent.To validate our approach, we repeated the synthesis of scaffold 51according to the literature. The samples prepared according to theliterature and according to the invention were identical based on TLC,mass spec, and NMR.

Example 2 Functionalization of Thiocarbamates to Make Chiral SubstitutedUrease, Semicarbazides and Carbazides and Potential Targets to BuildVariety of Peptidomimetics

Having in hands a robust method to generate these reactive intermediatesfrom acyl thiols using TCCA, like compounds mentioned in Example 1, thescope of the protocol was explored. Scheme 9 summarizes the results ofthe reactions that were performed:

At the beginning, thiocarbamate 59 was build from the L-phenylalaninemethyl ester 58 and the S-ethyl thiochloroformate in 90% yield, upontreatment 59 with TCCA/TBCl. The generation of the correspondingisocyanate, which can be isolated by simple filtration over a pad ofsilica to get rid of the other side products, was invisioned. Theresulting isocyanate was treated in situ with L-valinate amino acidt-butyl ester. The resulted urea was then isolated in 67% without anyepimerization. This experiment proved the efficacy of this technology toproduce chiral substituted urease and to link peptides from theN-terminus.

Knowing that the hydrazines are inferior nucleophiles compared totypical amines, the coupling protocol was modified, e.g., by changingthe base or the solvent. The isocyanate produced for thiocarbamate 59was isolated by simple filtration over a pad of silica, after thecomplete evaporation of the organic volatiles; the crude isocyanate wasdissolved in DMF and then treated with the appropriate hydrazinederivative in the presence of catalytic amount of DMAP. Semicarbazides61, 62, and 63 were collected in 39%, 70%, and 89% yields, respectively.

It is important to note that the reaction rates for the formation ofthese semicarbazides are much slower than those of the reverse couplingorder (see examples in Scheme 7) and that is because of the nature ofthe nucleophiles used. The reactions in scheme 9 are expected to besubstrate dependents especially the formation of tetra-substitutedderivatives like compounds 64-68. Reversing the addition order byinstalling the acylthiol on the hydrazine part followed by addition ofmore reactive amine may be one solution. However, this protocol may befurther developed to achieve a global solution for the formation of theessential carbazides (e.g., 70), which may be uses as synthons for thecreation of azatides peptidomimetics:

Example 3 Synthesis of Beta Peptides, and Other Bio Conjugates fromThioesters

The use of TCCA to generate, e.g., the reactive acyl chlorideintermediates, which facilitate the formation of the peptide bondwithout the need for expensive peptide coupling reagents, was tested.

To test this idea, inventors prepared α-amino acid thioesters and testedthe formation of the peptide bond under the protocol in Example 1. Thereaction rate of activation of thioesters using TCCA was found to bemuch faster than that for activation of thiocarbamates and thesemithiocarbazates. That is because of the neighboring nitrogen to thecarbonyl system. The reaction proceeded smoothly even at subzerotemperatures and without the need to add the TBACl. Careful NMR analysisconfirmed the formation of acyl chloride as a reactive intermediate toform the peptide bond, and this finding was further confirmed by theformation of the acyl chloride using different pathway by reacting theamino acid with SOCl₂ as a control experiment. Regardless of the highyields of the formation of the dipeptide, the reaction suffers acomplete epimerization of the alpha CH next to the thioester moiety.

Inventors repeated the experiment with beta amino acid thioesters tostudy the epimerization process and how to manage it in the light ofunderstanding the bond dissociation energies for the alpha CH in aminoacids and the captodative effect in stabilizing free radicals. The earlyresults showed that the TCCA protocol does not induce epimerization inbeta amino acids nor distant chiral centers other than the alpha CH. Theresults are summarized in Scheme 10:

These results are encouraging and show promise in exploring thisprotocol and technology in designing bioconjugates that can be easilycoupled to target proteins. The protocols could be used, e.g., inconjugating peptides, lipids, steroids or other active molecules thatcan be linked covalently to proteins.

Example 4

Unless otherwise mentioned, all starting materials were used directlyfrom the supplier without further purifications; all reactions werecarried out in oven-dried glassware using syringe and septa techniques,NMR spectra were collected on 500 or 600 MHz machines. Chemical shiftswere recorded relative to the deuterated solvent peak or the internalstandard tetramethylsilane (TMS) peak at (δ 0.00) and are reported inparts per million (ppm). Assignments for selected nuclei were determinedfrom 1H COSY and HSQC experiments. Low-resolution mass spectrometrymeasurements were collected on LTQ mass spectrometer using Xcalibursoftware. X-ray analysis were acquired using Bruker X8 Kappa Apex IIdiffractometer using Mo Kα radiation. The structure was solved usingdirect methods and standard difference map techniques, and was refinedby full-matrix least-squares procedures on F² with SHELXTL (Version2017/1). Thin-layer chromatography (TLC) was done on 0.25 mmthick-coated silica gel aluminum sheets. TLC plates were seen under UVlight with short and long wavelengths, or were observed after iodinestaining, or were visualized by heating the plates upon exposure to asolution of ammonium (VI) molybdate tetrahydrate and cerium (IV) sulfatetetrahydrate. In some cases KMnO₄ oxidation technique, and ninhydrinewere also applied to observe the TLC plates. Flash column chromatography(FCC) was implemented using silica gel 60 (230-400 mesh) and employed astepwise solvent polarity gradient, correlated with TLC mobility.

Procedure A: General Procedure to Prepare the Protected S-ethylHydrazinecarbothioate

To a solution of N-aminophthalimide (1000 mg, 6.1 mmol) in anhydrousdimethyl form amide (DMF) (10 mL) was added S-Ethyl chlorothioformate(600 uL, 6.1 mmol) and 4-dimethyl amino pyridine DMAP (150 mg, 0.61mmol). The reaction mixture was stirred at room temperature for 2 hours,at which a copious white precipitate was observed. The reaction mixturewas mixed with water (25 mL) and was extracted with EtOAc (25 mL×4). Thecombined organic layer was washed with brine (25 mL), dried over Na₂SO₄,filtered and evaporated under vacuum, and the residue was purified byFCC to give the protected S-ethyl hydrazine carbothioate

Procedure B: General Procedure to Synthesize the N-Substituted S-EthylHydrazinecarbothioates

To a solution of S-ethyl (1,3-dioxoisoindolin-2-yl) carbamothioate fromthe previous step (1 mmol) in anhydrous THF (10 mL) was added PPh₃ (1.5mmol) and alcohol (1.05 mmol). The stirred solution at 0° C. was treatedwith 40% DEAD solution in toluene drop-wise during 30 min (1.5 mmol).Then the reaction mixture was warmed up to r.t and stirred at thistemperature for 2 h. the reaction was stopped by evaporating the excessvolatiles in vacuum and the crude mixture was subjected to FCC to givethe corresponding alkyl substituent.

a) S-ethyl benzyl(1,3-dioxoisoindolin-2-yl)carbamothioate

A solution of S-ethyl (1,3-dioxoisoindolin-2-yl) carbamothioate (83 mg,0.332 mmol) in anhydrous THF (3.5 mL) was treated with benzyl alcohol(36 uL, 0.35 mmol), PPh₃ (130 mg, 0.5 mmol) and DEAD (215 uL, 0.5 mmol)according to above-mentioned procedure. After FCC purification thetitled compound was isolated as white crystalline material with (98 mg,87%).

Rf=(EtOAc/Hexane); ¹HNMR (600 MHz, CDCl₃) δ 7.78-7.71 (m, 4H), 7.25-7.13(m, 5H), 4.90 (s, 2H), 2.85 (m, 2H), 1.20 (m, 3H); ¹³C NMR (150 MHz,CDCl₃) δ 171.5, 164.9, 135.2, 134.0, 130.0, 128.7, 128.5, 124.3, 53.1,25.2, 14.9.

b) S-ethyl sec-butyl(1,3-dioxoisoindolin-2-yl) carbamothioate

A solution of S-ethyl (1,3-dioxoisoindolin-2-yl) carbamothioate (125 mg,0.5 mmol) in anhydrous THF (2.0 mL) was treated with R-2-butanol (69 uL,0.75 mmol), PPh₃ (197 mg, 0.75 mmol) and DEAD (330 uL, 0.75 mmol)according to above-mentioned procedure. After FCC purification thetitled compound was isolated as white crystalline material with (120 mg,78%). Rf=(EtOAc/Hexane); ¹HNMR (500 MHz, Acetoned₆) δ 8.04 (m, 4H), 4.58(q, J=6.8 Hz, 13.7 Hz, 1H), 2.83 (m, 2H), 1.77 (m, 1H), 1.47 (m, 1H),1.24-1.18 (m, 6H), 1.02 (t, J=7.3 Hz, 3H); ¹³C NMR (125 MHz, Acetoned₆)δ 170.3, 166.4, 136.4, 130.7, 124.8, 58.3, 28.6, 25.0, 17.6, 15.4, 11.6.

c) tert-butylN-(1,3-dioxoisoindolin-2-yl)-N-((ethylthio)carbonyl)glycinate(20180420-A659Y)

A solution of S-ethyl (1,3-dioxoisoindolin-2-yl) carbamothioate (125 mg,0.5 mmol) in anhydrous THF (2.0 mL) was treated with tert-butyl2-hydroxyacetate (99 mg, 0.75 mmol), PPh₃ (197 mg, 0.75 mmol) and DEAD(330 uL, 0.75 mmol) according to above-mentioned procedure. After FCCpurification the titled compound was isolated as white crystallinematerial with (171 mg, 93%). Rf=(EtOAc/Hexane); ¹HNMR (500 MHz,Acetoned₆) δ 8.05 (m, 4H), 4.44 (s, 2H), 2.88 (m, 2H), 1.45 (s, 9H),1.23 (m, 3H); ¹³C NMR (125 MHz, Acetoned₆) δ 171.6, 166.6, 164.8, 81,61.7, 52.2, 28.1, 25.2, 15.3.

d) S-ethyl(4-(benzyloxy)butyl)(1,3-dioxoisoindolin-2-yl)carbamothioate(20180309-A643Y)

A solution of S-ethyl (1,3-dioxoisoindolin-2-yl) carbamothioate (50 mg,0.2 mmol) in anhydrous THF (1.0 mL) was treated with 4-(benzyloxy)butan-1-ol (54 mg, 0.3 mmol), PPh₃ (78.6 mg, 0.3 mmol) and DEAD (132 uL,0.3 mmol) according to above-mentioned procedure. After FCC purificationthe titled compound was isolated as white crystalline material with (70mg, 85%). Rf=0.36 (20% EtOAc/Hexane); ¹HNMR (600 MHz, Acetoned₆) δ 7.87(m, 4H), 7.17 (m, 5H), 4.33 (s, 2H), 3.70 (t, J=6.42 Hz, 2H), 3.36 (t,J=5.82 Hz, 2H), 2.69 (q, J=7.32 Hz, 2H), 1.58 (m, 4H), 1.06 (t, J=7.32Hz, 3H); ¹³C NMR (150 MHz, Acetoned₆) δ 171.1, 165.6, 140.1, 136.4,130.7, 129.1, 128.3, 128.1, 127.9, 124.8, 73.2, 70.5, 50.3, 27.5, 25.5,25.1, 15.4.

e) tert-butyl3-((1,3-dioxoisoindolin-2-yl)((ethylthio)carbonyl)amino)propanoate(20180420-A654Y)

A solution of S-ethyl (1,3-dioxoisoindolin-2-yl) carbamothioate (125 mg,0.5 mmol) in anhydrous THF (2.0 mL) was treated with tert-butyl3-hydroxypropanoate (110 uL, 0.75 mmol), PPh₃ (197 mg, 0.75 mmol) andDEAD, (330 uL, 0.75 mmol) according to above-mentioned procedure. AfterFCC purification the titled compound was isolated as white crystallinematerial with (100 mg, 53%). Rf=(EtOAc/Hexane); ¹HNMR (600 MHz,Acetoned₆) δ 8.02 (m, 4H), 4.06 (t, J=6.7 Hz, 2H), 2.84 (m, 2H), 2.67(t, J=6.7 Hz, 2H), 1.23 (s, 9H), 1.21 (t, J=7.3 Hz, 3H); ¹³C NMR (150MHz, Acetoned₆) δ 170.3, 164.5, 135.4, 129.8, 123.9, 80.2, 44.2, 33.7,27.2, 24.2, 15.3.

f) S-ethyl (1,3-dioxoisoindolin-2-yl)(4-methoxybenzyl)carbamothioate(20180402-A644Y)

A solution, of S-ethyl (1,3-dioxoisoindolin-2-yl) carbamothioate (55 mg,0.25 mmol) in anhydrous THF (1.0 mL) was treated with p-methoxy benzylalcohol (45.5 mg, 0.33 mmol), PPh₃ (86.5 mg, 0.33 mmol) and DEAD (145uL, 0.33 mmol) according to above-mentioned procedure. After FCCpurification the titled compound was isolated as white crystallinematerial with (45 mg, 50%).

Rf=(EtOAc/Hexane); ¹HNMR (600 MHz, Acetoned₆) δ 7.82-7.78 (m, 4H), 7.15(m, 2H), 6.68 (m, 2H), 4.75 (s, 2H), 3.60 (s, 3H), 2.72 (m, 2H), 1.07(m, 3H); ¹³C NMR (150 MHz, Acetoned₆) δ 171.7, 165.1, 160.6, 136.4,135.1, 131.8, 130.4, 127.6, 124.8, 123.9, 114.5, 55.5, 53.3, 25.2, 15.4.

g) Benzyl(tert-butoxycarbonyl)(4-((1,3-dioxoisoindolin-2-yl)((ethylthio)carbonyl)amino)butyl)carbamate(20180420-A651W)

A solution of S-ethyl (1,3-dioxoisoindolin-2-yl) carbamothioate (55 mg,0.22 mmol) in anhydrous THF (1.0 mL) was treated with benzyl(4-hydroxybutyl) carbamatel (98.12 mg, 0.44 mmol), PPh₃ (115 mg, 0.44mmol) and DEAD (200 uL, 0.44 mmol) according to above-mentionedprocedure. After FCC purification to afford a clear film, this materialwas dissolved in DMF and treated with Boc anhydride (100 uL) and DMAP(10 mg) and left stirring at rt for 1 hr. then, the reaction mixture wasmixed with water and extracted with EtOAc (5 mL×3), the combined organiclayer was washed with brine, dried over Na₂SO₄, filtered, and evaporatedunder vacuum, after standard FCC purification using 40% EtOAc/Hexane,the compound was isolated as clear wax with (88 mg, 72.1%). Rf=0.36 (20%EtOAc/Hexane); ¹HNMR (600 MHz; Acetoned₆) δ 8.02 (m, 4H), 7.46-7.32 (m,5H), 5.19 (s, 2H), 3.83 (t, J=7.5 Hz, 2H), 3.68 (t, J=6.5 Hz, 2H), 2.84(m, 2H), 1.72-165 (m, 4H), 1.44 (s, 9H), 1.21 (t, J=7.3 Hz, 3H); ¹³C NMR(150 MHz, Acetoned₆) δ 171.1, 165.5, 154.7, 153.0, 137.2, 136.4, 135.2,130.7, 129.4, 129.0, 124.9, 82.9, 68.7, 50.2, 46.6, 28.2, 26.9, 25.8,25.2, 15.4.

h) S-ethyl (1,3-dioxoisoindolin-2-yl)(isobutyl) carbamothioate (A657)

A solution, of S-ethyl (1,3-dioxoisoindolin-2-yl) carbamothioate (125mg, 0.5 mmol) in anhydrous THF (2.0 mL) was treated with isobutanol (71uL, 0.75 mmol), PPh₃ (197 mg, 0.75 mmol) and DEAD (330 uL, 0.75 mmol)according to above-mentioned procedure. After FCC purification thetitled compound was isolated as white crystalline material with (150 mg,98%). Rf=(EtOAc/Hexane); ¹HNMR (600 MHz, Acetoned₆) δ.

i) The Boc Protected S-ethyl(1,3-dioxoisoindolin-2-yl)(3-(N,N′,N″tri-boc-guanidinopropyl)carbamothioate (20180402-A660W)

A solution of S-ethyl (1,3-dioxoisoindolin-2-yl) carbamothioate (125 mg,0.5 mmol) in anhydrous THF (2.0 mL) was treated with propandiol (57 uL,0.75 mmol), PPh₃ (197 mg, 0.75 mmol) and DEAD (330 uL, 0.75 mmol)according to above-mentioned procedure. The resulting alcohol was usedin the next step after FCC purification. A solution of the alcohol fromthe previous step (70 mg, 0.227 mmol) in anhydrous THF (1.0 mL) wastreated with N, N′, N″ tri-Boc-Guanidine (122 mg, 0.34 mmol), PPh₃ (89mg, 0.34 mmol) and DEAD (150 uL, 0.34 mmol) according toabove-mentioned. Upon purification using FCC protocol we collected (50mg, 35%).

j) S-ethyl((1H-indol-3-yl)methyl)(1,3-dioxoisoindolin-2-yl)carbamothioate(20181002-A648Y)

A solution of S-ethyl (1,3-dioxoisoindolin-2-yl) carbamothioate. (55 mg,0.22 mmol) in anhydrous THF (1.0 mL) was treated with (1H-indol-3-yl)methanol (97 mg, 0.66 mmol), PPh₃ (173 mg, 0.66 mmol) and DEAD (290 uL,0.66 mmol) according to above-mentioned procedure. After FCCpurification the titled compound was isolated as yellow amorphousmaterial with (75 mg, 90%). Rf=(EtOAc/Hexane); ¹HNMR (600 MHz,Acetoned₆) δ 10.16 (bs, 1H), 7.93-7.85 (m, 4H), 7.62 (d, J=7.9 Hz, 1H),7.33 (m, 2H), 7.07 (m, 1H), 6.99 (t, J=7.6 Hz, 1H), 5.17 (s, 2H), 2.90(m, 2H), 1.24 (m, 3H); ¹³C NMR (150 MHz, Acetoned₆) δ 171, 164.1, 136.6,135.3, 129.4, 126.1, 123.7, 121.5, 118.6, 111.3, 108.0, 44, 24, 15.

k) S-ethyl (1,3-dioxoisoindolin-2-yl)(2-(methylthio)ethyl)carbamothioate(20180309-A642Y)

A solution of S-ethyl (1,3-dioxoisoindolin-2-yl) carbamothioate (60 mg,0.24 mmol) in anhydrous THF (1.0 mL) was treated with 2-(methylthio)ethan-1-ol (31 uL, 0.36 mmol), PPh₃ (94.3 mg, 0.36 mmol) and DEAD (160uL, 0.36 mmol) according to above-mentioned procedure. After FCCpurification the titled compound was isolated with (55 mg, 90%).Rf=(EtOAc/Hexane); ¹HNMR (600 MHz, Acetoned₆) δ 7.95 (bs, 4H), 3.92 (t,J=7.8 Hz, 2H), 2.81-2.71 (m, 4H), 2.00 (s, 3H), 1.47 (t, J=7.32 Hz, 3H);¹³C NMR (150 MHz, Acetoned₆) δ 171.1, 165.5, 136.3, 130.8, 124.8, 49.9,31.9, 25.1, 15.3, 15.2.

l) S-ethyl (1,3-dioxoisoindolin-2-yl)(isopropyl) carbamothioate (A648W)

A solution of S-ethyl (1,3-dioxoisoindolin-2-yl) carbamothioate (55 mg,0.22 mmol) in anhydrous THF (1.0 mL) was treated with 2-propanol (35 uL,0.44 mmol), PPh₃ (115 mg, 0:44 mmol) and DEAD (200 uL, 0.44 mmol)according to above-mentioned procedure. After FCC purification thetitled compound was isolated with (55 mg, 86%). Rf=(EtOAc/Hexane); ¹HNMR(600 MHz, Acetoned₆) δ.

m) S-ethyl (1,3-dioxoisoindolin-2-yl)(methyl)carbamothioate (A652W)

A solution of S-ethyl (1,3-dioxoisoindolin-2-yl) carbamothioate (125 mg,0.5 mmol) in anhydrous THF (2.0 mL) was treated with methanol (24 uL,0.75 mmol), PPh₃ (197 mg, 0.75 mmol) and DEAD (326 uL, 0.75 mmol)according to above-mentioned procedure. After FCC purification thetitled compound was isolated with (140 mg, 99%). Rf=(EtOAc/Hexane);¹HNMR (600 MHz, Acetoned₆) S.

Procedure C: Procedure to prepare the Aza-serine amine-acid analogs(this procedure can be applied to the reaction of the hydrazones withaldehydes in general.)

((1,3-dioxoisoindolin-2-yl)((ethylthio) carbonyl)amino)methyl Acetate(20180420-A645W-OAc)

A solution of S-ethyl (1,3-dioxoisoindolin-2-yl) carbamothioate (55 mg,0.22 mmol) in anhydrous Toluene (2.0 mL) was treated withparaformaldehyde (60 mg, 2.2 mmol), the reaction mixture was heated at80° C. until the disappearance of the starting material. The isolatedcrude was dissolved in EtOAc and treated with Ac₂O and DMAP. After FCCpurification, the titled compound was isolated as clear crystals.Rf=(EtOAc/Hexane); ¹HNMR (500 MHz, Acetoned₆) δ 7.91 (m, 4H), 5.53 (s,2H), 2.74 (q, J=14.4, 7.1 Hz, 2H), 1.91 (s, 3H), 1.09 (t, J=7.25, 3H);¹³C NMR (125 MHz, Acetoned₆) δ 171.7, 171.3, 164.9, 136.6, 130.4, 125.1,69.7, 25.3, 20.6, 15.1.

Procedure D: General Procedure to Prepare the Tert-butyl 2-((ethylthio)carbonyl)hydrazine-1-carboxylate

To a solution of tert-butyl carbazate (1320 mg, 10.0 mmol) in anhydrousdiethyl ether (40 mL) was added S-Ethyl chlorothioformate (1050 uL, 10.0mmol) and pyridine (890 uL, 11:2 mmol). The reaction mixture was stirredat r:t for 45 min, at which a copious white precipitate was observed.The reaction mixture was mixed with water (25 mL) and was extracted withEtOAc (25 mL×4). The combined organic layer was washed with brine (25mL), dried over Na₂SO₄, filtered and evaporated under vacuum, and theresidue was purified by FCC to give the protected S-ethyl hydrazinecarbothioate

Tert-butyl 2-benzyl-2-((ethylthio)carbonyl)hydrazine-1-carboxylate

To a solution of the Boc-protected S-ethyl hydrazine carbothioate (200mg, 0.9 mmol) in anhydrous THF (2.0 mL) was added PPh₃ (357 mg, 1.36mmol) and benzyl alcohol (107 uL, 0.99 mmol) and DEAD (616 uL, 1.36mmol) according to above-mentioned procedure. The residue was purifiedby FCC to give the titled compound as colorless crystals (214 mg, 69%).Rf=(EtOAc/Hexane); ¹HNMR (600 MHz, Acetoned₆) δ 8.60 (bs, 1H), 7.32 (m,5H), 4.76 (ABq, J=12.2 Hz, Δδ=0.99 ppm, 2H), 2.83 (m, 2H), 1.25 (t,J=7.32 Hz, 3H); ¹³C NMR (150 MHz, Acetoned₆) 820181002-A738).

Procedure E: Procedure to Prepare the Aza-Proline Analog (this Procedureis Applicable to the Synthesis of Alkyl Substituted Hydrazine CarbamatesUsing NAH and Alkyl Halides) Tert-butyl2-((ethylthio)carbonyl)pyrazolidine-1-carboxylate

To a solution of Boc-protected S-ethyl hydrazine carbothioate (210 mg,0.95 mmol) in anhydrous DMF (5.0 mL) was added NaH (84.0 mg, 2.1 mmol).The reaction mixture was stirred at 0° C. for 15 min, at whichdiiodopropane (110 uL, 0.95 mmol) solution in DMF (1.0 mL) was addeddrop-wise. The reaction mixture was stirred at room temperature for 16hours. Upon completion, the reaction was treated with water (25 mL) andwas extracted with EtOAc (25 mL×4). The combined organic layer waswashed with brine (25 mL), dried over Na₂SO₄, filtered and evaporatedunder vacuum, and the residue was purified by FCC to give the titledcompound with (85 mg, 34%). ¹HNMR (500 MHz, Acetoned₆) δ 4.00 (m, 2H),3.15 (m, 1H), 3.04 (m, 1H), 2.88 (m, 2H), 2.06 (m, 2H), 1.46 (s, 9H),1.23 (t, J=7.32 Hz, 3H); ¹³C NMR (125 MHz, Acetoned₆) δ 175.2, 156.9,82.6, 46.8, 46.2, 28.3, 25.7, 24.4, 15.2. (20180830-A733),

Procedure F: General Procedure to Prepare the Substituted Semicarbazides

To a solution of the thiocarbamate (0.1 mmol) in DCM (2.0 mL) was addedTBACl (0.1 mmol) and TCCA (0.1 mmol). The reaction mixture was stirredat r.t for 15 min then cooled down to 0° C., using ice bath. Theice-cold reaction mixture was added amine (0.15 mmol) anddiisopropylethylamine (DIPEA) (0.4 mmol). The reaction was left stirringat room temperature for another 30 min or until the disappearance of thestarting materials. Upon completion, the reaction mixture was treatedwith saturated solution of sodium thiosulfate and stirred for 15-30 min,and then the aqueous layer was extracted with organic solvent (×3times). The combined organic layers was washed with brine, dried overNa₂SO₄, filtered, and evaporated under vacuum. The resulting residue wassubjected to standard FCC using EtOAc/Heaxane with gradient increase ofpolarity. The titled compounds were characterized by mass spectroscopyand NMR.

a) Tert-butyl (benzyl(1,3-dioxoisoindolin-2-yl)carbamoyl)valinate

To a solution of the thiocarbamate (34 mg, 0.1 mmol) in DCM (1.0 mL) wasadded TBACl (28 mg, 0.1 mmol) and TCCA (23 mg, 0.1 mmol). The reactionmixture was stirred at room temperature for 15 min then cooled down to0° C. using ice bath. The ice-cold reaction mixture was added amine(0.15 mmol) and diisopropylethylamine (DIPEA) (0.4 mmol). The reactionmixture was processed according to procedure F to give the titledcompound with (43 mg, 95%). ¹HNMR (500 MHz, Acetoned₆) δ 7.85 (m, 4H),7.27 (m, 2H), 7.23 (m, 3H), 6.42 (d, J=10.0 Hz, 1H), 4.93 (s, 2H), 4.19(q, J=5.0, 10 Hz, 1H), 1.98 (m, 1H), 1.44 (s, 9H), 0.91 (d, J=5.0 Hz,3H), 0.88 (d, J=5.0 Hz, 3H); ¹³C NMR (125 MHz, Acetoned₆) δ 171.7,166.1, 157.2, 137.2, 135.6, 131.3, 131.1, 129.9, 129.0, 128.5, 124.2,81.5, 60.8, 53.5, 31.6, 28.2, 19.51, 18.6. (20181011-A758).

b) Methyl (benzyl(1,3-dioxoisoindolin-2-yl)carbamoyl)prolinate(20180226-A635W) in Note Book is A638Y

To a solution of the thiocarbamate (15 mg, 0.044 mmol) in DCM (1.0 mL)was added TBACl (12 mg, 0.095 mmol) and TCCA (10 mg, 0.095 mmol). Thereaction mixture was stirred at r.t for 15 min then cooled down to 0° C.using ice bath. The ice-cold reaction mixture was added amine (9.0 mg,0.066 mmol) and diisopropylethylamine (DIPEA) (32 uL, 0.176 mmol). Thereaction mixture was processed according to procedure F to give thetitled compound with (15 mg, 84%). ¹HNMR (600 MHz, CDCl₃) δ 7.68 (m,4H), 7.29 (m, 2H), 7.14 (m, 3H), 4.75 (ABq, J=14.2 Hz, Δδ=0.18 ppm, 2H),4.42 (q, J=7.9, 4.9 Hz, 1H), 3.55 (s, 3H), 3.30-3.21 (m, 2H), 2.07 (m,1H), 1.85 (m, 1H), 1.75 (m, 2H); ¹³C NMR (150 MHz, CDCl₃) δ 173.0,165.4, 165.1, 158.4, 135.0, 134.9, 130.0, 129.4, 128.5, 128.2, 124.1,124.0, 61.0, 54.5, 52.3, 48.4, 29.6, 25.1.

c) MethylO-benzyl-N-(benzyl(1,3-dioxoisoindolin-2-yl)carbamoyl)serylprolinate.(20180510-A666W)

To a solution of the thiocarbamate (34 mg, 0.1 mmol) in DCM (1.0 mL) wasadded TBACl (28 mg, 0.1 mmol) and TCCA (23 mg, 0.1 mmol). The reactionmixture was stirred at room temperature for 15 min then cooled down to0° C. using ice bath. The ice-cold reaction mixture was added amine(0.15 mmol) and diisopropylethylamine (DIPEA) (0.4 mmol). The reactionmixture was processed according to procedure F to give the titledcompound with (mg, %). ¹HNMR (600 MHz, Acetoned₆) δ 7.74-7.69 (m, 4H),7.287.05 (m, 10H), 6.60 (bd, J=8.04 Hz, 1H), 4.81-4.74 (m, 3H), 4.39(ABq, J=12.06 Hz, Δδ=0.02 ppm, 2H), 4.20 (q, J=8.6, 4.6 Hz, 1H), 3.69(m, 1H), 3.61 (m, 2H), 148 (m, 2H), 3.46 (s, 3H), 3.44 (m, 2H), 2.09 (m,2H), 1.88 (m, 2H), 1.76 (m, 1H); ¹³C NMR (150 MHz, Acetoned₆) δ 173.1,169.6, 166.1, 166.0, 157.0, 139.7, 137.2, 135.7, 131.2, 130.1, 129.2,129, 128.5, 128.4, 128.2, 124.3, 124.3, 73.8, 73.6, 75.5, 72.0, 70.9,62.0, 59.9, 53.4, 513, 53.0, 52.7, 52.1, 47.7, 47.1, 44.0, 25.6.

d) Ethyl (benzyl(1,3-dioxoisoindolin-2-yl)carbamoyl)glycinate(20180226-A639W)

The reaction mixture was processed according to procedure F on 0.025mmol scale. This reaction gave the titled compound with (9.0 mg, 94%).¹HNMR (600 MHz, CDCl₃) δ 7.83 (m, 2H), 7.76 (m, 2H), 7.28 (m, 2H), 7.24(m, 4H), 5.29 (t, J=4.68 Hz, 1H), 4.92 (s, 2H), 4.16 (q, J=7.14 Hz, 2H),4.02 (d, J=4.92 Hz, 2H), 1.26 (t, J=7.14 Hz, 3H).

e) Protected Aza-Lysine-Glycine-Ethyl Ester Dipeptide (20180420-A651Y)

The reaction mixture was processed according to procedure F on 0.025mmol scale. This reaction gave the titled compound with (%). ¹HNMR (600MHz, CDCl₃) δ 7.79 (m, 4H), 7.31-7.19 (m, 5H), 6.86 (m, 1H), 5.03 (s,2H), 3.94 (q, J=7.15 Hz, 2H), 3.67 (m, 2H), 3.61 (t, J=7.25 Hz; 2H),3.51 (t, J=6.7 Hz, 2H), 1.92-145 (m, 4H), 1.28 (s, 9H), 1.07 (t, J=7.55Hz, 3H); ¹³C NMR (125 MHz, Acetoned₆) δ 170.8, 166.4, 157.3, 154.7,152.9, 137.1, 135.7, 131.5, 129.3, 129.0, 124.3, 82.8, 68.7, 61.2, 49.3,46.7, 42.9, 28.9, 26.9, 26.1, 14.5.

f) Tert-butylN-(1,3-dioxoisoindolin-2-yl)-N-((2-ethoxy-2-oxoethyl)carbamoyl)glycinate(20180420-A660Y)

The reaction mixture was processed according to procedure F on 0.025mmol scale. This reaction gave the titled compound with (%). ¹HNMR (600MHz, CDCl₃) δ 7.85-7.74 (m, 4H), 5.68 (bs, 1H), 4.28 (s, 2H), 4.10 (q,J=7.14 Hz, 2H), 3.93 (d, J=4.9 Hz, 2H), 1.38 (s, 9H), 1.88 (t, J=7.2 Hz,3H); ¹³C NMR (150 MHz, CDCl₃) δ 167.6, 165.0, 155.7, 135.2, 130.0,124.4, 82.9, 61.8, 51.8, 43.0, 28.1, 14.3.

g) Ethyl((4-(benzyloxy)butyl)(1,3-dioxoisoindolin-2-yl)carbamoyl)glycinate(20180309-A644W)

The reaction mixture was processed according to procedure F on 0.025mmol scale. This reaction gave the titled compound with (%). ¹HNMR (600MHz, CDCl₃) δ 7.81 (m, 2H), 7.72 (m, 2H), 7.24 (m, 5H), 5.63 (bs, 1H),4.41 (s, 2H), 4.11 (t, 2H), 3.71 (m, 4H), 3.51 (t, J=5.9 Hz, 2H), 1.75(m, 2H), 1.62 (m, 2H), 1.18 (t, J=7.3 Hz, 3H); ¹³C NMR (150 MHz, CDCl₃)δ 170.4, 165.9, 156.1, 138.3, 135.0, 130.1, 128.7, 128.0, 127.9, 124.2,73.5, 61.5, 49.7, 42.7, 26.0, 14.3.

h) Tert-butyl2-benzyl-2-((1-(tert-butoxy)-3-methyl-1-oxobutan-2-yl)carbamoyl)hydrazine-1-carboxylate.(2018002-A750)

This compound was prepared according to procedure F on 0.12 mmol scale.This reaction gave the titled compound with (45 mg, 89%). ¹HNMR (600MHz, Acetoned₆) δ 7.3× (m, 5H), 6.05 (m, 1H), 4.24 (m, 1H), 2.89 (m,2H), 2.06 (m, 1H), 1.53 (m, 18H), 0.90 (d, 3H), 0.88 (d, 3H.

f) Tert-butyl2-((1-(tert-butoxy)-3-methyl-1-oxobutan-2-yl)carbamoyl)pyrazolidine-1-carboxylate.(20181002-A752)

This compound was prepared according to procedure F on 0.058 mmol scale.This reaction gave the titled compound with (15 mg, 69%). ¹HNMR (600MHz, Acetoned₆) δ 6.0× (d, 1H), 4.0× (q, 1H), 3.7× (m, 2H), 3.0× (m,2H), 2.0× (m, 1H), 2.06 (m, 1H), 1.93 (m, 2H), 1.28 (m, 18H), 0.9× (d,3H), 0.7× (d, 3H);

j) Methyl (benzyl(1,3-dioxoisoindolin-2-yl)carbamoyl)phenylalaninate(20181101-A763)

This compound was prepared according to procedure F on 0.095 mmol scale.This reaction gave the titled compound with (35 mg, 82%). ¹HNMR (600MHz, Acetoned₆) δ 7.73-7.769 (m, 4H), 7.23-7.04 (m, 10H), 6.66 (m, 1H),4.72 (ABq, J=14.8 Hz, 2H), 4.44 (m, 1H), 3.48 (m, 3H), 2.87 (dd, J=13.7,5.8 Hz, 1H), 2.2.75 (m, 1H); ¹³C NMR (150 MHz, Acetoned₆) δ 173.0,166.0, 165.9, 157.0, 138.2, 137.1, 135.7, 131.2, 130.3, 130.0, 129.2,129.0, 128.5, 127.5, 124.3, 124.2, 56.4, 53.3, 52.2, 38.3.

k) Methyl (benzyl(1,3-dioxoisoindolin-2-yl)carbamoyl)tryptophanate(20181101-A767)

This compound was prepared according to procedure F on 0.1 mmol scale.This reaction gave the titled compound with (48 mg, 87%). ¹HNMR (500MHz, Acetoned₆) δ 10.05 (bs, 1H), 7.88-7.81 (m, 4H), 7.51 (d, J=7.9 Hz,1H), 7.34 (m, 3H), 7.21 (m, 3H), 7.11 (m, 1H), 7.04 (t, J=7.15 Hz, 1H),6.96 (t, J=7.3, 1H), 6.74 (m, 1H), 4.86 (ABq, J=14.8 Hz, 2H), 4.65 (m,1H), 3.60 (s, 3H), 3.19 (m, 1H), 3.05 (m, 1H); ¹³C NMR (125 MHz,Acetoned₆) δ 173.4, 166.0, 157.1, 137.5, 137.1, 135.7, 131.1, 130.0,129.0, 128.5, 124.6, 124.3, 124.2, 122.2, 119.7, 119.0, 112.2, 110.8,55.8, 53.3, 52.5, 28.2.

l) methylN²-(benzyl(1,3-dioxoisoindolin-2-yl)carbamoyl)-N^(ω)-((2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl)argininate.(20181101-A768)

This compound was prepared according to procedure F on 0.1 mmol scale.This reaction gave the titled compound with (52 mg, 71%). ¹HNMR (600MHz, Acetoned₆) δ 7.85-7.81 (m, 4H), 7.37 (d, J=6.8 Hz, 2H), 7.24-7.19(m, 3H), 6.95 (m, 1H), 6.47 (m, 2H), 4.88 (ABq, J=14.6 Hz, 2H), 4.36 (m,1H), 3.62 (s, 3H), 3.17 (m, 2H), 2.99 (s, 6H), 2.82 (s, 3H), 2.56 (s,3H), 2.49 (s, 3H) 1.76 (m, 1H), (1.58-1.1.48 (m, 3H), 1.44 (s, 6H); ¹³CNMR (150 MHz, Acetoned₆) δ 173.5, 166.1, 165.9, 159.0, 157.4, 138.8,137.1, 135.7, 132.9, 131.3, 131.2, 129.0, 128.5, 125.4, 124.3, 124.2,117.5, 87.0, 53.2, 52.3, 43.7, 19.5, 18.2, 12.6.

m) Tert-butyl (benzyl(1,3-dioxoisoindolin-2-yl)carbamoyl)leucinate(20181120-A769)

This compound was prepared according to procedure F on 0.1 mmol scale.This reaction gave the titled compound with (42 mg, 90.3%). ¹HNMR (600MHz, Acetoned₆) δ 7.85-7.81 (m, 4H), 7.39-7.18 (m, 5H), 6.74 (d, J=8.16Hz, 1H), 4.90 (s, 2H), 4.32 (m, 1H), 1.72 (m, 1H), 1.43 (m, 11H), 0.89(d, J=6.54 Hz, 3H), 0.85 (d, J=6.66 Hz, 3H); ¹³C NMR (150 MHz,Acetoned₆) δ 172.9, 166.1, 166.0, 157.3, 137.3, 135.6, 131.3, 130.1,129.0, 128.4, 124.2, 124.2, 81.2, 53.9, 53.1, 41.4, 28.2, 25.3, 23.4,21.9.

n) Methyl (benzyl(1,3-dioxoisoindolin-2-yl)carbamoyl)-L-threoninate(20181120-A774)

This compound was prepared according to procedure F on 0.05 mmol scale:This reaction gave the titled compound with (18 mg, 88%). ¹HNMR (500MHz, Acetoned₆) δ7.90-7.84 (m, 4H), 7.43 (m, 2H), 7.27-7.21 (m, 3H),6.37 (d, J=8.45 Hz, 1H), 4.93 (ABq, J=14.9 Hz, 2H), 4.41 (dd, J=3.25,8.9 Hz, 1H), 4.19 (m, 1H), 3.79 (d, J=6.0 Hz, 1H), 3.64 (s, 3H), 1.50(d, J=6.4 Hz, 3H); ¹³C NMR (125 MHz, Acetoned₆) δ 172.0, 166.1, 166.0,157.6, 68.2, 60.4, 53.6, 52.3, 14.6.

o) 4-benzyl 1-methyl(benzyl(1,3-dioxoisoindolin-2-yl)carbamoyl)-L-aspartate (20181120-A777)

This compound was prepared according to procedure F on 0.05 mmol scale.This reaction gave the titled compound with (21 mg, 82%). ¹HNMR (500MHz, Acetoned₆) δ 7.8×-7.8× (m, 4H), 7.42-7.15 (m, 10H), 5.13 (q, J=xxHz, 2H), 4.89 (q, J=xx Hz, 2H), 4.81 (m, 1H), 3.57 (s, 3H), 2.88 (m,1H), 2.70 (dd, J=6.8, 16.25 Hz, 1H); ¹³C NMR (125 MHz, Acetoned₆) δ171.5, 171.4, 165.8, 157.1, 137.1, 137.0, 135.7, 131.1, 130.0, 129.3,129.0, 128.8, 128.7, 128.5, 124.3, 67.3, 53.2, 52.0, 51.5, 36.9.

p) Methyl (benzyl(1,3-dioxoisoindolin-2-yl)carbamoyl)-L-methioninate(20181120-A770)

This compound was prepared according to procedure F on 0.1 mmol scale.This reaction gave the titled compound with (42 mg, 95%). ¹HNMR (600MHz, Acetoned₆) δ 7.88-7.82 (m, 4H), 7.39-7.18 (m, 5H), 6.99 (d, J=8.4Hz, 1H), 4.89 (q, J=14.7 Hz, 2H), 4.56 (m, 1H), 3.65 (s, 3H), 2.5745 (m,2H), 2.02 (s, 3H), 2.00-1.97 (m, 1H), 1.82-1.78 (m, 1H); ¹³C NMR (150MHz, Acetoned₆) δ 173.4, 166.0, 165.9, 157.3, 137.1, 135.7, 131.1,130.1, 129.0, 128.5, 124.3, 124.2, 53.7, 53.1, 52.3, 32.1, 30.9, 15.3.

q) methylN²-(benzyl(1,3-dioxoisoindolin-2-yl)carbamoyl)-N⁶-(tert-butoxycarbonyl)-L-lysinate(20181203-A785)

This compound was prepared according to procedure F on 0.076 mmol scale.This reaction gave the titled compound with (35 mg, 86%). ¹HNMR (400MHz, Acetoned₆) δ 7.83-7.81 (m, 4H), 7.40-7.19 (m, 5H), 6.89 (d, J=8.0Hz, 1H), 5.89 (bs, 1H), 4.89 (ABq, J=16.0 Hz, 2H), 4.38 (m, 1H), 3.64(s, 3H), 3.03 (m, 2H), 1.73 (m, 1H), 1.43 (m, 14H); ¹³C NMR.

Example 5 Procedures for Solution Phase and Solid Phase PeptideSynthesis

General

Solid phase peptide syntheses (SPPSes) were executed using Tribute®Peptide synthesizer from Gyros Protein Technologies, Inc. The machine isfully automated with two independent reaction vessels withpolytetrafluoroethylene frits, five solvent positions and 101 amino acidpositions. Preloaded Fmoc-L-Arg(Pbf) Wang resin (200-400 mesh, 0.3mmol/g) and Rink amide MBHA resin (0.3 mmol/g), and premixed Fmoc aminoacids with HATU coupling agent were purchased from Pure Pep™/GyrosProtein Technologies, Inc. All solvents, and reagents used in synthesisare peptide-grade and were purchased from Gyros Protein Technologies.The solvents and reagents were used without further treatment or drying.The Aza-amino acid were synthesized in Applicant's labs and integratedin the synthesis with minimal interruption to the automation, i.e, theactivated aza-amino acid residue were added manually, by interruptingthe synthesis momentarily, then the rest of the steps including thecleavage, washing, and drying were maintained automated. Analyses wereperformed using Water's technology HPLC equipped with a 1525 binarypump, and the use of analytical column (Phenomenex kinetex 2.6 μm EVOC18 analytical column 100Å 150×4.6 mm). Chromatography was performed atambient temperature with flow rate of 1.0 mL/min with linear gradientfrom Water (0.05% TFA): CH₃CN (0.05% TFA) [95:5] to Water Water (0.05%TFA): CH₃CN (0.05% TFA) [5:95] and resolved peaks were detected by 2998photodiode Array (PDA) Detector at 254 and/or 215 nm and characterizedby low resolution mass spectrometry instrument (Thermo ScientificLTQXL™) with ESI ion-source and positive mode ionization. Purificationof the all peptidomimetics were performed on preparative HPLCpurification system (Waters Prep 150 LC system combining 2545 BinaryGradient Module using XSelect Peptide CSH C18 OBD Prep Column, 130A, 5μm, 19 mm×150 mm. Chromatography was performed at ambient temperaturewith a flow rate of 18 mL/min with a linear gradient from Water (0.1%FA): CH₃CN (0.1% FA)[95:5] to Water (0.1% FA): CH₃CN (0.1% TFA) [5:95]in 12 minutes, monitored by 2998 Photodiode Array (PDA) Detector UV at254 nm and/or 215. NMR spectra were recorded in acetone-d6, CDCl₃, D2O,and DMSO-d6 with TMS for 1H (500 and/or 600 MHz) and 13C (125 and/or 150MHz) as an internal reference.

General Procedure for SPPS

All solid phase peptide couplings were performed at ambient temperatureusing Tribute® Peptide synthesizer from Gyros Protein Technologies, Incfollowing a protocol that included:

-   -   1. Swelling: The resin (loaded with the first Fmoc-protected        amino acid) was swelled twice successively for 20 minutes in        DMF, each swelling step followed by drain and drying step.    -   2. Fmoc Cleavage: the protected amino acid/or peptide was shaken        for 2.0 min with 20% piperidine solution in DMF to remove the        Fmoc group. The process was repeated twice, followed by several        washing steps with DMF (3-5 times).    -   3. Amino acid coupling: 5 equivalents of the next acylating        component (Fmoc protected amino acid or activated Aza-amino        acid), 5 equivalents of Hexafluorophosphate Azabenzotriazole        Tetramethyl Uronium coupling reagent (“HATU”), and 10        equivalents of N-methylmorpholine (base) were used to add the        next amino acid in the sequence. This step is fully automated        and was run according to the software installed on the Tribute®        synthesizer. The amino acid with coupling reagent were delivered        to the reaction vessel from the specified loading position upon        dissolution. Then the base was added as 0.4 M solution in DMF,        the total volume of solvent was adjusted to give 0.2 M solution.        The coupling time was limited to 15 minutes shacking followed by        draining, washing steps. Step 2 and step 3 were repeated until        the sequence synthesis was done.    -   4. Washing: repeated washing steps were performed after each        cleavage or coupling event using MDF as solvent (2-3 times). At        the final coupling or cleavage steps additional washing with DCM        was performed (5-6 times) to remove any trace of DMF. The        process usually followed by drying step.    -   5. Cleavage from the Resin: 5.0 mL of a freshly made solution of        TFA/H₂O/TIPS (95:2.5:2.5. v/v/v) was cooled down to 0° C. and        added at OC to a 0.3 mmol of Resin. The mixture was shaken for 2        hours, then was filtered, the remaining resin was further washed        with 0.5-1.0 mL of TFA/H2O) (95:5, v/v) solution. The filtrate        was precipitated by adding 10 mL of 1:1 solution of        ether:hexane. Upon centrifugation, the resulting solid was        dissolved in a 1:1 solution of CH₃CN:H₂O. The resulting solution        was lyophilized.    -   6. Purification: Purification of the all peptidomimetics were        performed on preparative HPLC purification system (Waters Prep        150 LC system combining 2545 Binary Gradient Module using        XSelect Peptide CSH C18 OBD Prep Column, 130A, 5 μm, 19        mm×150 mm. Chromatography was performed at ambient temperature        with a flow rate of 18 mL/min with a linear gradient from Water        (0.1% FA): CH₃CN (0.1% FA)[95:5] to Water (0.1% FA): CH₃CN (0.1%        TFA) [5:95] in 12 minutes, monitored by 2998 Photodiode Array        (PDA) Detector UV at 254 nm and/or 215.

Example 6 Synthesis of Aza-Amino Acid Surrogates

To a solution of the thiocarbazate (110 mg, 0.5 mmol) in anhydrous THF(1.0 mL) PBu₃ (187 uL, 0.75 mmol) and isobutanol (47 uL, 0.5 mmol). Thereaction mixture was cooled down to 0° C., then a 40% solution of DEAD(327 uL, 0.75 mmol) was added dropwise over 30 min. upon completion ofthe addition, the reaction mixture was allowed to rt gradually (30 min).Then, the volatiles were removed under vacuum and the residue waspurified by FFC and the use of gradient of hexanes/ether. The targetcompound was collected as colorless crystals (81 mg, 59%); ¹HNMR (500MHz, Acetoned₆) δ 8.63 (bs, 1H), 3.66 (m, 1H), 3.09 (m, 1H), 2.76 (m,2H), 1.93 (m, 2H), 1.20 (t, J=6.15 Hz, 3H), 0.9 (bd, 6H). ¹³C NMR (125MHz, Acetoned₆) δ 172.9, 154.7, 81.3, 56.8, 28.5, 27.3, 24.7, 20.5,20.3, 15.7. structure was confirmed by X-ray.

To a solution of the thiocarbazate (220 mg, 1.0 mmol) in anhydrous THF(1.0 mL), PBu₃ (303 uL, 1.2 mmol) and R-2-butanol (200 uL, 2.0 mmol).The reaction mixture was cooled down to 0° C., then a 40% solution ofDEAD (522 uL, 1.2 mmol) was added dropwise over 30 min. upon completionof the addition, the reaction mixture was allowed to warm up to roomtemperature gradually (30 min). Then, the volatiles were removed undervacuum and the residue was purified by FFC and the use of gradient ofhexanes/ether. The target compound was collected as colorless crystals(195 mg, 70%); ¹HNMR (500 MHz, Acetoned₆) structure was confirmed byX-ray.

To a solution of the thiocarbazate (110 mg, 0.5 mmol) in anhydrous THF(1.0 mL) PBu₃ (187 uL, 0.75 mmol) and tert-butyl 2-hydroxyacetate (99mg, 0.75 mmol). The reaction mixture was cooled down to 0° C., then a40% solution of DEAD (300 uL, 1.36 mmol) was added dropwise over 30minutes. Upon completion of the addition, the reaction mixture wasallowed to warm up to room temperature gradually (30 minutes). Then, thevolatiles were removed under vacuum and the residue was purified by FFCand the use of gradient of hexanes/ether. Rf=0.44 (20% ether/hexane).The target compound was collected as colorless crystals (138 mg, 83%);¹HNMR (500 MHz, Acetoned₆) δ 8.51 (bs, 1H), 4.69 (m, 1H), 3.73 (m, 1H),2.79 (m, 2H), 1.45 (m, 18H), 1.22 (t, J=6.15 Hz, 3H). ¹³C NMR (125 MHz,Acetoned₆) δ 173.1, 82.4, 81.8, 51.5, 28.4, 28.2, 24.8, 15.6. Structurewas analyzed by X-ray.

To a solution of the thiocarbazate (60 mg, 0.272 mmol) in anhydrous THF(0.5 mL) PBu₃ (83 uL, 0.3.27 mmol) and benzyl (4-hydroxybutyl)carbamate(73 mg, 0.327 mmol). The reaction mixture was cooled down to 0° C., thena 40% solution of DEAD (327 uL, 0.75 mmol) was added dropwise over 30minutes. Upon completion of the addition, the reaction mixture wasallowed to warm up to room temperature gradually (30 minutes). Then, thevolatiles were removed under vacuum and the residue was purified by FFCand the use of gradient of hexanes/ether. The target compound wascollected as colorless wax (110 mg, 90%); ¹HNMR (500 MHz, Acetoned₆) δ8.66 (s, 1H), 7.36-7.29 (m, 5H), 6.31 (bs, 1H), 5.05 (s, 2H), 3.86 (m,1H), 3.31 (m, 1H), 3.16 (m, 2H), 2.74 (m, 2H), 1.57 (m, 2H), 1.54 (m,2H), 1.46 (m, 9H), 1.20 (t, J=6.15 Hz, 3H). ¹³C NMR (125 MHz, Acetoned₆)δ 172.6, 157.3, 154.9, 138.6, 129.2, 128.7, 128.6, 81.4, 66.4, 49.0,41.2, 28.4, 27.9, 25.0, 24.7, 15.7.

To a solution of the thiocarbazate (220 mg, 1.0 mmol) in anhydrous THF(1.5 mL) PBu₃ (379 uL, 1.5 mmol) and N-Boc indole-3-carbinol (309 mg,1.25 mmol). The reaction mixture was cooled down to 0° C., then a 40%solution of DEAD (650 uL, 1.5 mmol) was added dropwise over 30 minutes.Upon completion of the addition, the reaction mixture was allowed towarm up to room temperature gradually (30 min). Then, the volatiles wereremoved under vacuum and the residue was purified by FFC and the use ofgradient of hexanes/ether. The target compound was collected ascolorless gummy material (380 mg, 85%); ¹HNMR (500 MHz, Acetoned₆) δ

To a solution of the thiocarbazate (165 mg, 0.75 mmol) in anhydrous THE(1.0 mL) PBu₃ (284 uL, 1.125 mmol) and 2-Hydroxyethyl methyl sulfide (92mg, 0.8 mmol). The reaction mixture was cooled down to 0° C., then a 40%solution of DEAD (489 uL, 1.125 mmol) was added dropwise over 30minutes. Upon completion of the addition, the reaction mixture wasallowed to warm up to room temperature gradually (30 minutes). Then, thevolatiles were removed under vacuum and the residue was purified by FFCand the use of gradient of hexanes/ether. The target compound wascollected as colorless crystals (150 mg, 68%); ¹HNMR (500 MHz,Acetoned₆)

To a solution of the thiocarbazate (160 mg, 0.73 mmol) in anhydrous THF(1.5 mL) PBu₃ (193 uL, 0.73 mmol) and N-Phthalimido-1-propanol (150 mg,0.73 mmol). The reaction mixture was cooled down to 0° C., then a 40%solution of DEAD (660 uL, 1.0 mmol) was added dropwise over 30 minutes.Upon completion of the addition, the reaction mixture was allowed towarm up to room temperature gradually (30 minutes). Then, the volatileswere removed under vacuum and the residue was purified by FFC and theuse of gradient of hexanes/ether. The target compound was collected ascolorless gummy material (125 mg, 42%); ¹HNMR (500 MHz, Acetoned₆)

To a solution of semicarbazate A822 (75 mg, 0.18 mmol) in EtOH (2.0 mL)was added Hydrazine (23 uL, 0.37 mmol). The reaction mixture was heatedat 40° C. for 2 hours, at which the starting material completelydisappeared based on tlc. The organic volatiles were removed undervacuum and the residue was purified using C18 column with gradient ofWater/CH₃CN. The pure fraction was collected based on the massspectroscopy analysis. After 16 hour lyophilizing, the resulting aminewas subjected to the next step without further treatment. To a solutionof the resulting amine the previous step (23.0 mg, 0.08 mmol) in DCM(2.0 mL) was added Cbz-protected methylcarbamothioate (86:0 mg, 0.24mmol), DIPEA (15 uL, 0.08 mmol) and one crystal of DMAP. The reactionmixture was stirred at room temperature for 16 hours. Then, the organicvolatiles were removed under vacuum and the residue was purified onsilica and the use of gradient of EtOAc/Hexane to give 50 mg (45% overtwo steps).

To a solution of the thiocarbazate (220 mg, 1.0 mmol) in anhydrous THF(2.0 mL) PBu₃ (500 uL, 2.0 mmol) and3-(Di-tert-butyloxycarbonyl)guanidinopropanol (375 mg, 1.18 mmol). Thereaction mixture was cooled down to 0° C. Then, a 40% solution of DEAD(870 uL, 2.0 mmol) was added dropwise over 30 minutes. Upon completionof the addition, the reaction mixture was allowed to warm up to roomtemperature gradually (30 min). Then, the volatiles were removed undervacuum and the residue was purified by FFC and the use of gradient ofhexanes/ether. The target compound was collected as colorless gummymaterial (225 mg, 43%); ¹HNMR (500 MHz, Acetoned₆) S.

To a solution of S-ethyl 1-benzyl hydrazine-1-carbothioate (304 mg, 1.45mmol) in DMF (8.0 mL) was added N-Boc Proline (343 mg, 0.1.59 mmol),HATU (722.4 mg, 1.59 mmol), HOBt (214 mg, 1.59 mmol), and N-methylmorpholine NMM (650 uL, 6.4 mmol). The reaction mixture was stirred atroom temperature for 20 hours, then was treated with 0.5 M citric acidsolution. The aqueous layer was extracted with EtOAc (15.0 mL×3), thecombined organic fractions were washed with saturated NaHCO₃ solution,brain, and cold water, the organic layer was dried over Na₂SO₄,filtered, and evaporated under vacuum. The cured material was purifiedover silica by the use of gradient solvent mixture of Hexanes and EtOAc;the purified fraction was dried under high vacuum to give a whitecrystalline material (248 mg, 42%) LRMS (ESI, MNa⁺) m/z calc forC₂₀H₂₉N₃O₄SNa⁺ 430.18, found 430.3.

To a solution of phthalimidyl-S-ethyl ethanethioate (62.25 mg, 0.25mmol) in DCM (5.0 mL) was added TBACl (71.0 mg, 0.25 mmol). The reactionmixture was stirred at 0° C. for one min; then, the reaction mixture wastreated with TCCA (58.0 mg, 0.25 mmol). The reaction mixture was allowedto warm up to room temperature in 10 minutes. At this time, a solutionof thiocarbazate (Azaphe thioate) (52.0 mg, 0.25 mmol), DIPEA (177.0 uL,1.0 mmol) and one crystal of DMAP in DCM (0.5 mL) was added at 0° C.,and the reaction was stirred at room temperature for 2.0 hours before itwas stopped by the addition of sodium thiosulfate (1 mL). The organiclayer was washed with a saturated solution of sodium hydrogen sulfate,saturated solution of sodium bicarbonate, and brine. The organic layerwas dried over sodium sulfate, filtered, and evaporated under vacuum.The residue was purified using FCC and gradient of hexane/ethyl acetateto give a gummy material (55 mg, 55% yield).

To a solution of S-ethyl hydrazinecarbothioate (240 mg, 2.0 mmol) in DMF(10.0 mL) was added N-alphaBoc-N-delta phthaloyl-L-Ornithine acid (800mg, 2.0 mmol), HATU (760.5 mg, 2.0 mmol), HOBt (270 mg, 2.0 mmol), andDIPEA (1.4 mL, 4.0 mmol). The reaction mixture was stirred at roomtemperature for 20 hours, then was treated with 0.5 M citric acidsolution. The aqueous layer was extracted with EtOAc (15.0 mL×3), thecombined organic fractions were washed with saturated NaHCO₃ solution,brain, and cold water, the organic layer was dried over Na₂SO₄,filtered, and evaporated under vacuum. The cured material was purifiedover silica by the use of gradient solvent mixture of Hexanes and EtOAc;the purified fraction was dried under high vacuum to give a white solidmaterial (400 mg, 40%).

To a solution of S-propyl chlorothioformate (500 uL, 3.9 mmol) in THF (5mL) was added DMAP (480 mg, 3.9 mmol) at 0° C. The reaction mixture wasstirred at 0° C. until complete dissolution of the DMAP crystals and theappearance of copious white precipitate. At this point, a solution ofFmocNHNH₂ (1.0 g, 3.9 mmol) in DCM (5 mL) was added slowly. Upon thecompletion of the addition, the reaction mixture was left to warm up toroom temperature gradually. The reaction progress was monitored by TLC;after 2 hours, the reaction was stopped by adding ethyl ether (50 mL),the organic phase was transferred into a separatory funnel. It wastreated with 1 M solution of HCl (25 mL). The organic phase was washedwith a saturated solution of NaHCO₃ (20 mL), Braine (20 mL), dried overNa₂SO₄, and filtered. The excess solvent was evaporated under vacuum,and the crude mixture was suspended in 5 mL of CH₃CN, then filtered anddried to give 1.25 g of white non-crystalline powder (yield 89%), whichwas used in the next step without further purification. LRMS (ESI, MNa⁺)m/z calc for C₁₉H₂₀N₂NaO₃S, 379.11, found 379.3.

To a solution of thiocarbazate A9130 (356 mg, 1.0 mmol) in CHCl₃ (3.70mL) was added PBu₃ (379 uL, 1.5 mmol) and BnOH (108 uL, 1.0 mmol) at−10° C. (ice/acetone/Salt). To the reaction mixture was added slowlyduring one hour a 20% solution of DEAD (1.3 mL, 1.5 mmol). The reactionmixture was left stirring at the same temperature for an additional houror until the complete disappearance of the thiocarbazate. The reactionwas stopped by adding 1 M solution of HCl (5.0 mL), and diethyl ether(10 mL). The organic layer was washed successively with a saturatedsolution of NaHCO₃ and brine. Later, the organic layer was dried overNa₂SO₄, filtered, and evaporated under vacuum. The crude material wassubjected to FCC using gradient solution of ether and hexanes. Thepurified weigh 321 mg (72% yield). HPLC analysis indicated that thepurified fraction is a mixture of 5:1 of two Regio-isomers, A9164T andA9164B. The purified fraction was suspended in 1 mL of CH₃CN at roomtemperature then filtered; the purification process was repeated twiceto give solely A9164T as a pure compound based on the HPLC analysis.LRMS (ESI, MNa+) m/z calc for C₂₆H₂₆N₂O₃SNa 469.2, found 469.3.

To a solution of thiosemicarbazide (1100 mg, 5.0 mmol) in anhydrous THF(20.0 mL) was added Barton Base (1.0 mL, 5.0 mmol). The reaction mixturewas stirred at 0° C. for 10 min, at which diiodopropane (650 uL, 0.95mmol) solution in THF (5.0 mL) was added drop-wise (over 30 minutes).The reaction mixture was stirred at 0° C. for 1 hour, then was left towarm up to room temperature gradually. Upon completion, the reaction wastreated with 0.5 M HCl (10 mL) and was extracted with EtOAc (25 mL×4).The combined organic layer was washed with saturated NaHCO₃ (25 mL),Brine (25 mL), dried over Na₂SO₄, filtered and evaporated under vacuum,and the residue was purified by FCC to give the titled compound with(1.25 g, 96%). ¹HNMR (500 MHz, Acetoned₆) δ 4.00 (m, 2H), 3.15 (m, 1H),(m, 1H), 2.88 (m, 2H), 2.06 (m, 2H), 1.46 (s, 9H), 1.23 (t, J=7.32 Hz,3H); ¹³C NMR (125 MHz, Acetoned₆) δ 175.2, 156.9, 82.6, 46.8, 46.2,28.3, 25.7, 24.4, 15.2. LRMS (ESI, MNa+) m/z calc for C₁₁H₂₀N₂O₃SNa283.11, found 283.1. A9188.

To a solution of the thiocarbazate A701 (260 mg, 1.0 mmol) in DCM (4.0mL) was added 1.0 mL of TFA. The reaction mixture was stirred at roomtemperature for 30 minutes; then, the volatiles was evaporated invacuum. The residue was dried further under a high vacuum for anadditional 30 min. Later, the residue was dissolved in anhydrous THF(5.0 mL) and treated with solid NaHCO₃. (840 mg, 10 mmol). Thesuspension was treated with Fmoc-Cl (258 mg, 1.0 mmol), and the reactionmixture was stirred at room temperature for 2 hours. The NaHCO₃ wasfiltered the filtrate was evaporated to dryness then was purified usingFCC and gradient of ether/hexanes. LRMS (ESI, MNa+) m/z calc forC₂₁H₂₂N₂O₃SNa 405.12, found 405.3.

To a solution of thiocarbazate A9130 (356 mg, 1.0 mmol) in CHCl₃ (3.70mL) was added PBu₃ (379 uL, 1.5 mmol) and3-(Di-tert-butyloxycarbonyl)guanidinopropanolii (317 mg, 1.0 mmol) at−10° C. (ice/acetone/Salt). To the reaction mixture was added slowly a20% solution of DEAD (1.3 mL, 1.5 mmol) during one hour. The reactionmixture was left stirring at the same temperature for an additional houror until the complete disappearance of the thiocarbazate. The reactionwas stopped by adding 1 M solution of HCl, 5.0 mL, and diethyl ether 10mL. The organic layer was washed successively with a saturated solutionof NaHCO₃ and brine. Later, the organic layer was dried over Na₂SO₄,filtered, and evaporated under vacuum. The crude material was subjectedto FCC using gradient solution of ether and hexanes. The purifiedmaterial weigh 350 mg (42% yield) or 80% based on recoveredthiocarbazate A9130. LRMS (ESI, MH+) m/z calc for C₃₃H₄₅N₅O₇SH⁺656.31,found 656.3.

To a solution of thiocarbazate A9130 (356 mg, 1.0 mmol) in CHCl₃ (4.5mL) was added PBu₃ (379 uL, 1.5 mmol) and tert-butyl 2-hydroxy acetate(132 mg, 1.0 mmol) at −10° C. (ice/acetone/Salt). A 20% solution of DEAD(1.3 mL, 1.5 mmol) was added dropwise to the reaction mixture duringone-hour using a syringe pump with an addition rate of 1.3 mL/h. Thereaction mixture was left stirring at the same temperature for anadditional hour or until the complete disappearance of the startingmaterial. The volatile solvents were removed under vacuum, and theresidue was loaded into a silica column and eluted with gradient solventof Hexane/EtOAc up to 20% EtOAc. LRMS (ESI, MH+) m/z calc forC25H30N2O5SNa+493.18, found 493.2.

Example 7 Coupling Aza-Amino Acids Surrogates with Native Amino Acids

To a solution of thiocarbazate A783 (28.0 mg, 0.1 mmol) in DCM (1.0 mL)was added TBACl (28 mg, 0.1 mmol). The reaction mixture was stirred at0° C. for one min; then, the reaction mixture was treated with TCCA (23mg, 0.1 mmol). The reaction mixture was allowed to warm up to roomtemperature in 15 minutes. At this time, a solution of phenylalaninemethyl ester (32.3 mg, 0.15 mmol) and DIPEA (71 uL, 0.4 mmol) in DCM(0.5 mL) was added at 0° C., and the reaction was stirred at roomtemperature for 30 minutes before it was stopped by the addition ofsodium thiosulfate (1 mL). The organic layer was washed with a saturatedsolution of sodium hydrogen sulfate, saturated solution of sodiumbicarbonate, and brine. The organic layer was dried over sodium sulfate,filtered, and evaporated under vacuum. The residue was purified usingFCC and gradient of hexane/ethyl acetate to give a white solid material6).

To a solution of thiocarbazate A790 (22.0 mg, 0.065 mmol) in DCM (1.0mL) was added TBACl (18.24 mg, 0.065 mmol). The reaction mixture wasstirred at 0° C. for one minute; then, the reaction mixture was treatedwith TCCA (15 mg, 0.065 mmol). The reaction mixture was allowed to warmup to room temperature in 15 minutes. At this time, a solution ofphenylalanine methyl ester (21.5 mg, 0.1 mmol) and DIPEA (46 uL, 0.26mmol) in DCM (0.5 mL) was added at 0° C., and the reaction was stirredat room temperature for 30 minutes before it was stopped by the additionof sodium thiosulfate (1 mL). The organic layer was washed with asaturated solution of sodium hydrogen sulfate, saturated solution ofsodium bicarbonate, and brine. The organic layer was dried over sodiumsulfate, filtered, and evaporated under vacuum. The residue was purifiedusing FCC and gradient of hexane/ethyl acetate to give a white solidmaterial (25.5 mg, 87% yield). Rf=0.375 (30% EtOAc/Hexane). ¹HNMR (500MHz, Acetoned₆) δ 1.90 (bs, 1H), 7.32-7.20 (m, 5H), 6.35 (m, 1H), 4.60(m, 1H), 3.66 (s, 3H), 3.09 (m, 2H) 2.8 (s, 2H), 1.44 (m, 18H). ¹³C NMR(125 MHz, Acetoned₆) δ.

To a solution of thiocarbazate A738 (310.0 mg, 1.0 mmol) in DCM (18.0mL) was added TBACl (277 mg, 1.0 mmol). The reaction mixture was stirredat 0° C. for one minute; then, the reaction mixture was treated withTCCA (232, 1.0 mmol). The reaction mixture was allowed to warm up toroom temperature in 15 minutes. At this time, a solution of Arginine(Pbf) methyl ester (715.5 mg, 1.5 mmol) and DIPEA (750 uL, 4.0 mmol) inDCM (2.0 mL) was added at 0° C., and the reaction was stirred at roomtemperature for 30 minutes before it was stopped by the addition ofsodium thiosulfate (5.0 mL). The organic layer was washed with asaturated solution of sodium hydrogen sulfate, saturated solution ofsodium bicarbonate, and brine. The organic layer was dried over sodiumsulfate, filtered, and evaporated under vacuum. The residue was purifiedusing FCC and gradient of hexane/ethyl acetate to give a White solidmaterial. (330 mg, 48% yield). LRMS (ESI, MH+) m/z calc forC₃₃H₄₈N₆O₈SH⁺689.33, found 689.3.

Azapeptide A8163 (95.0 mg, 0.138 mmol) was treated with 4M HCl (2.0 mL)at 0° C. the reaction was stirred for one hour with gradual warm up toroom temperature. Upon completion based on TLC and mass spec, the excesssolvent was evaporated under vacuum, and the residue was dried furtherunder high vacuum. Later, the deprotected Aza peptide (81.0 mg, 0.138)was dissolved in DMF (1.0 mL) and then was treated with acid A8192 (7.0mg, 0.138 mmol), HATU (53.0 mg, 0.138 mmol), HOBt (19.0 mg, 0.138 mmol),and DIPEA (103 uL, 0.56 mmol). The reaction mixture was stirred at roomtemperature for 16 hours; then, the reaction was treated with asaturated solution of sodium hydrogen sulfate (2.0 mL) The aqueous layerwas extracted with DCM (2.0 mL×4). The combined organic layer was washedwith the brain, dried over sodium sulfate, filtered, and evaporatedunder vacuum, the resulting residue was purified using silica gel andgradient of EtOAc/MeOH, up to 5% MeOH. The purified fraction wasassessed by HPLC. LRMS (ESI, MH+) m/z talc for C₆₄H₈₅N₁₁O₁₄SH⁺1264.60,found 12264.6.

Azapeptide A8163 (81.0 mg, 0.1177 mmol) was treated with 4M HCl (2.0 mL)at 0° C. the reaction was stirred for one h with gradual warm up to roomtemperature. Upon completion based on TLC and mass spec (mass found589.26), the excess solvent was evaporated under vacuum, and the residuewas dried further under high vacuum. Later, the deprotected Aza peptide(69.0 mg, 0.1177) was dissolved in DMF (1.0 mL) and then was treatedwith acid A8166 (130 mg, 0.107 mmol), HATU (45.0 mg, 0.1177 mmol), HOBt(16.0 mg, 0.1177 mmol), and NMM (79 uL, 0.43 mmol). The reaction mixturewas stirred at room temperature for 16 hours; then, the reaction wastreated with a saturated solution of sodium hydrogen sulfate (2.0 mL).The aqueous layer was extracted with DCM (2.0 mL×4). The combinedorganic layer was washed with the brain, dried over sodium sulfate,filtered, and evaporated under vacuum, the resulting residue waspurified using silica gel and gradient of EtOAc/MeOH, up to 5% MeOH. Thepurified fraction was assessed by HPLC. LRMS (ESI, MH+) m/z talc forC₈₈H₁₂₀N₁₆O₁₉S2H⁺1769.84, found 1769.8.

To a solution of thiocarbazate A992 (20.0 mg, 0.05 mmol) in DCM (1.0 mL)was added TBACl (14.0 mg, 0.05 mmol). The reaction mixture was stirredat 0° C. for one minute; then, the reaction mixture was treated withTCCA (12 mg, 0.05 mmol). The reaction mixture was allowed to warm up toroom temperature in 15 minutes. At this time, a solution of the freeamine tripeptide (SPF) (34.0 mg, 0.075 mmol) and DIPEA (35 uL, 0.2 mmol)in DCM (1.0 mL) was added at 0° C., and the reaction was stirred at roomtemperature for 30 minutes before it was stopped by the addition ofsodium thiosulfate (5.0 mL). The organic layer was washed with asaturated solution of sodium hydrogen sulfate, saturated solution ofsodium bicarbonate, and brine. The organic layer was dried over sodiumsulfate, filtered, and evaporated under vacuum. The residue was purifiedusing FCC and gradient of hexane/ethyl acetate to give a white foamymaterial. LRMS (ESI, MNa+) m/z calc for C₄₃H₄₄N₆O₉H⁺789.32, found 789.3.

Example 8 Integration of the Azaphenylalanine in SPPS

Wang Arginine-loaded Resin® was swelled in DMF for 20 minutes, followedby treatment with a 20% solution of Piperidine in DMF for 2 min usingtwo cycles to cleave the Fmoc group. After successive washes with DMF,the resin was suspended in DMF mixed with 10 equivalents of the N-Methylmorpholine base (NMM) for 10 minutes. During this time, and separately,4 equivalents of the thiocarbazate A9164T in 2 mL of CH₃CN, then wastreated with an equal molar ratio of TBACl, after shacking the mixturefor 1 minute, equal molar ratio of TCCA was added to activate thethiocarbazate scaffold: The reaction mixture was stirred at roomtemperature for 5 min, then was centrifuged. The clear supernatant wasintroduced into the suspended resin and the pH value was adjusted to 8.0by adding additional 10 equivalents of NMM (neat). The Reaction mixturewas shaken for 15 minutes, followed by 5 cycles of washing then, drying.The resulting dipeptide was treated with acetic anhydride (Ac₂O) andDiisopropylethylamine (DIPEA) to cap any left arginine (two cycles). Asmall amount of the resin was cleaved using TFA/DCM mixture, and theresulting peptide was analyzed by HPLC, which indicated a completeconversion and absence of capped products. LRMS (ESI, MH+) m/z calc forC₂₉H₃₂N₆O₅H, 545.25, found 545.4.

Example 9 Functionalization of Aza-Peptide with Native Amino Acid

1. Preparation of the Fmoc-Pro(Cl): To a solution of Fmoc-Proline (1.0g, 3.0 mmol) in DCM (10 mL) was added 0.1 mL of DMF. At 0° C., thereaction mixture was treated with thionyl chloride (2.0 mL). Thereaction mixture was brought gradually to room temperature and leftstirring for an additional 2 h. The volatiles was removed under vacuum,and the residue was triturated with a solution of ether:hexane (1:1,v/v) (10 mL). The residue was dried under high vacuum for 30 min andused in the next step without further treatment.

2. Cleavage of the Fmoc group from the dipeptide that bound to theresin: Dipeptide A19171 (34 mg, 0.01 mmol) was suspended in 250 uL ofDMF. The mixture was then agitated for 10 minutes, followed by removalof the solvent by filtration; then the process was repeated one moretime. The swelled resin was treated with 50% solution of morpholine inDMF (250 uL), the mixture was shaken for 2 min, then the solvent wasdrained, and the process was repeated one more time. The Fmoc cleavageprocess was followed by several washes with DMF (250 uL×3). Theresulting resin-bound peptide from the previous step (approx. 0.01 mmol)was suspended in dioxane (200 uL), treated with NaHCO₃ (16.8 mg, 0.2mmol), and shaken for 10 minutes. Fmoc-Pro(Cl) from the first step (18.0mg, 0.05 mmol) was dissolved in dioxane (200 uL) and introduced to theSPPS reaction vessel containing the NaHCO₃ in Dioxane. The resin wasshaken at ambient temperature for 45 min. Then, the solvent was drained,and the resin was washed successively with H₂O (300 uL×2), DMF (300uL×3), and DCM (300 uL×5). The resin was dried, and a small sample wascleaved with TFA for analysis.

Example 10

Using SPPS and following the above-mentioned protocol, 2-azabradykinin(“2AzaBK”), 5-azabradykinin (“5AzaBK”), 8-azabradykinin (“8AzaBK”),7-azabradykinin (“7AzaBK”); and 2,8azabradykin (“2,8AzaBK”) wereproduced. The chemical formulas for 2AzaBK, 8AzaBK, 7AzaBK, 2,8AzaBK arefollows:

The yield and purity of the synthesized compounds is provided in Table1.

TABLE 1 AzaBK T_(R)* Purity Mass Entry code Min HPLC Found 1 2AzaBK4.891   97% 1060.56 2 5AzaBK 7.45    70% 1282.62 Crude 3 7AzaBK 5.06692.6% 1060.56 4 8AzaBK 5.072 97.7% 1060.56 5 2,8AzaBK 4.940 98.5%1061.55 *Tr = retention time which is the time when the peak of thecorresponding peptide eluite from the C18 chromatography column duringthe HPLC analysis.*Tr=retention time which is the time when the peak of the correspondingpeptide eluate from the C18 chromatography column during the HPLCanalysis.

In the preceding specification, the invention has been described withreference to specific exemplary embodiments and examples thereof. Itwill, however, be evident that various modifications and changes may bemade thereto without departing from the broader spirit and scope of theinvention as set forth in the claims that follow. The specification anddrawings are accordingly to be regarded in an illustrative manner ratherthan a restrictive sense. All documents cited herein, as well as textappearing in the figures, are hereby incorporated by reference in theirentirety for all purposes to the same extent as if each were soindividually denoted.

What is claimed is:
 1. A compound of the Formula (I):

wherein A is phthalimidyl, Y is a bond, X is NR₅, D is an alkyl, and R₅is a side chain radical of an amino acid.
 2. The compound of claim 1,wherein R₅ is the side chain radical of an amino acid selected from thegroup consisting of alanine, histidine, glutamic acid, tryptophan,valine, leucine, lysine, methionine, tyrosine, isoleucine, arginine,glycine, asparagine, serine, threonine, cysteine, and glutamine.
 3. Thecompound of claim 1, which is of the formula

wherein R₅ is a side chain a side chain radical of an amino acid.
 4. Thecompound of claim 3, wherein R₅ is the side chain radical of an aminoacid selected from the group consisting of alanine, histidine, glutamicacid, tryptophan, valine, leucine, lysine, methionine, tyrosine,isoleucine, arginine, glycine, asparagine, serine, threonine, cysteine,and glutamine.
 5. The compound of claim 3, which is


6. A process for preparing a compound according to claim 1 comprisingforming the compound of claim 1 by reacting a semi-protected hydrazinewith a chlorothioformate to form an S-alkylthiocarbazate, and alkylatingthe resulting S-alkylthiocarbazate selectively via Mitsunobu reaction, adirect alkylation with NaH or with an alkyl halide.
 7. A process forsynthesizing an aza-peptide comprising activating a thioester with TCCAto form a reactive chloride, and coupling the reactive chloride with anamine, wherein the thioester is a compound according to claim
 1. 8. Theprocess of claim 7, wherein the aza-peptide is a compound of Formula(XVI):

wherein

is at the N-terminus and/or the C-terminus and/or at or adjacent to acleavage and/or a hydrolysis site of the compound of Formula (XVI); B isselected from the group consisting of hydrogen, —NH₂, —CONH₂, —COOR₁₉,—COH, —COC₁-C₄ alkyl, —COC₁-C₄ haloalkyl, —OH, an amino acid, an azaamino acid, a 2 to 60-mer peptide, a 2 to 60-mer aza peptide, and a 2 to60-mer azatide; D is selected from the group consisting of —NH₂, —NNH₂,—NHCOCH₃, —NHCH₃, —N(CH₃)₂, —CONH₂, —COOH, —COH, —COC₁-C₄ alkyl,—COC₁-C₄ haloalkyl, an amino acid, an aza amino acid, a 2 to 60-merpeptide, a 2 to 60-mer aza peptide, and a 2 to 60-mer azatide; R₁₉ isselected from the group consisting of C₁-C₆ alkyl, methoxyl, ethoxyl,propoxyl, a C₁-C₆ haloalkyl and a protecting group; and R is selectedfrom the group consisting of side chain radicals of aspartic acid,phenylalanine, alanine, histidine, glutamic acid, tryptophan, valine,leucine, lysine, methionine, tyrosine, isoleucine, arginine, glycine,asparagine, serine, threonine, cysteine, and glutamine.